Discovery of 3-(Indol-5-yl)-indazole Derivatives as Novel Myeloid

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Discovery of 3-(indol-5-yl)-indazole derivatives as novel myeloid differentiation protein 2/toll-like receptor 4 antagonists for treatment of acute lung injury Zhiguo Liu, Lingfeng Chen, Pengtian Yu, Yali Zhang, Bo Fang, Chao Wu, Wu Luo, Xianxin Chen, Chenglong Li, and Guang Liang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00316 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 18, 2019

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Journal of Medicinal Chemistry

Discovery

of

3-(indol-5-yl)-indazole

derivatives

as

novel

myeloid

differentiation protein 2/toll-like receptor 4 antagonists for treatment of acute lung injury

Zhiguo Liua,#,*, Lingfeng Chenb,#, Pengtian Yua,c, Yali Zhanga, Bo Fanga, Chao Wua, Wu Luoa, Xianxin Chena, Chenglong Lia, Guang Lianga,b,*

a

Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical

University, Wenzhou, Zhejiang 325035, China b

School of Chemical Engineering, Nanjing University of Science and Technology,

Nanjing, Jiangsu, 210094, China c

Department of Pharmacy, The First Affiliated Hospital, College of Medicine,

Zhejiang University, Zhejiang Hangzhou 310000, China # These

authors contributed equally to this work.

* Corresponding author: Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University. 1210 University Town, Wenzhou, Zhejiang 325035, China Tel: (+86)-577-86699892; Fax: (+86)-577-86699892. E-mail: [email protected]

Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University. 1210 University Town, Wenzhou, Zhejiang 325035, China Tel: (+86)-577-86699396; Fax: (+86)-577-86699396. E-mail: [email protected]

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Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Acute lung injury (ALI) is often caused by systemic inflammatory responses. Targeting myeloid differentiation protein 2/toll-like receptor 4 (MD2-TLR4) complex may be a promising way to treat Gram-negative bacterial-induced inflammatory disorders. In this study, we report the design and synthesis of a new series of 3-(indol-5-yl)-indazoles, which were evaluated for their anti-inflammatory activities in macrophages. Among the analogues generated, the promising 3-(indol-5-yl)-indazole analogue 22m inhibited lipopolysaccharides (LPS)-induced expression of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) in macrophages with IC50 values of 0.89 and 0.53 μM, respectively. Compound 22m was then identified as MD2-TLR4 antagonist in suppressing LPS-induced inflammatory responses. In vivo administration of 22m significantly inhibited macrophage infiltration and ameliorated histopathological changes in lung tissues of LPS-challenged mice. Our studies have identified a new 3-(indol-5-yl)-indazole, 22m, as potent MD2-TLR4 inhibitor and lay the groundwork for the future drug development of anti-inflammatory agents for the treatment of ALI.

Keywords: 3-(indol-5-yl)-indazole, acute lung injury, inflammation, myeloid differentiation protein 2, toll-like receptor 4


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INTRODUCTION Acute lung injury (ALI) and acute respiratory distress syndrome are characterized by pulmonary infiltrates, hypoxemia, and injury to both the vascular endothelium and lung alveolar epithelium.1,

2

ALI and acute respiratory distress syndrome commonly result from

sepsis, trauma, and acute pancreatitis. While systemic and direct lung injury may initiate the process, expansion of inflammatory response is responsible for injury progression. Macrophages account for approximately 95% of airspace leukocytes and are critically involved in the development of ALI following infection and non-infectious stimuli by producing proinflammatory cytokines.3 Among these cytokines, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) contribute most to the deterioration seen in ALI.4 Elevated levels of both IL-6 and TNF-α have been found in human and experimental ALI.5, 6 Experimentally, ALI has been produced in preclinical models through administration of bacterial endotoxin.7 Lipopolysaccharide (LPS), the main element of Gram-negative bacteria, plays an important role in acute inflammatory diseases such ALI and sepsis.8 Toll-like receptor 4 (TLR4) is one of the pattern recognition receptors that recognizes pathogen-associated molecular patterns, including LPS, lipooligosaccharide (LOS), and lipid A from bacteria.9 Studies have shown that LPS activates the TLR4 signaling pathway through an indispensable accessory protein called the myeloid differentiation protein 2 (MD2). MD2 associates with TLR4 to form (LPS-MD2-TLR4)2 complex on cell membrane, leading to the recruitment of adaptor protein, myeloid differentiating primary response gene 88 (MyD88). The dimerization of MyD88 activates pro-inflammatory signal transduction through the downstream intercellular kinase cascades and transcriptional factors, which up-regulate the



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expression of pro-inflammatory cytokines.10, 11 Therefore, targeting MD2-TLR4 complex may be a promising way to treat ALI, sepsis, and other acute inflammatory diseases.12, 13 Several compounds with anti-inflammatory activities have been reported to target MD2 or

TLR4 (Figure 1). Natural products such as 1 (Curcumin),14 2 (Xanthohumol)15 and 3 (Caffeic acid phenethyl ester) are reported as potential modulators of TLR4 signaling owing to their ability to interact with MD2.16 The triterpenoids celastrol and asiatic acid are also active in disrupting TLR4 signaling, experimental binding studies showed these two compounds compete with LPS for MD-2 binding.17 However, these natural compounds may interact with multiple targets in addition to MD2-TLR4. In addition, E. coli lipid A mimetics were developed to modulate the interaction of LPS with MD2/TLR4 complex. A synthetic phospholipid analogue 5 (PE-DTPA)18 improved the survival rate of LPS-challenged C57BL/6 mice in a dose-dependent manner. Cationic amphiphile (6, IAXO-102)19 based on monosaccharide scaffolds efficiently inhibited TLR4 signaling by competition from LPS for binding to MD-2. The most famous lipid A mimetic is tetra-acylated lipid A (7, Eritoran), which directly binding to the hydrophobic pocket of MD2.20,

21

Compound 8 (TAK-242)

selectively inhibits TLR4 signal by irreversibly binding to the Cys thiol of TLR4, thereby forming a covalent adduct with TLR4.22 Yin and co-workers23 also developed β-amino alcohol derivative 4 targeting TLR4 as a potential antiseptic compound. However, among all MD2-TLR4 antagonists so far developed, only antagonists 7 and 8 reached clinical trials, but unfortunately, both failed to reduce mortality among patients with severe sepsis in Phase 3 studies.24, 25 Therefore, development of an effective MD2/TLR4 inhibitor with new structural scaffold is highly desirable.


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Figure 1. Structures of natural and synthetic MD2/TLR4 antagonists 1-8.

Indole and indazole have wide biological applications and they are potentially valuable as building-blocks in medicinal chemistry.26,

27

In recent years, some of the indazole and

indole derivatives, such as 9 (5-aminoindazole), 10 (benzidamine), and 11 (indomethacin), have been applied to the treatment of acute or chronic inflammation as well as a variety of inflammatory diseases (Figure 2).28-30 However, to the best of our knowledge, these drugs share many of the potential adverse effects, including burning sensation, headache, itching, erythema,31 upset stomach, skin rash, or renal function disorders.32 As part of our ongoing anti-inflammatory drug discovery activities, we are engaging to find lead compounds with new structural skeleton for the treatment of ALI. Here, we envisioned that the introduction of



an indole moiety to the 5-aminoindazole scaffold might yield a novel series of potent anti-inflammatory agents. In addition, we found that a large number of natural and synthetic compounds, such as 12 (LY2874455),33 13 (1,3,5-tris(2-hydroxyethyl)isocyanurate)34 and FDA approved 14 (Dasatinib),35 with therapeutic effects on cancer and related diseases, contain one N-hydroxyethyl group (Figure 2). Evidences suggest that the hydroxyethyl group may contribute to the biological activity of these compounds, since it is the attachment point of the pyrophosphate radicle in cocarboxylase.36 We further try to see if linking hydroxyethyl group at indole N-atom enhances the anti-inflammatory ability. Hence, we report here the design, synthesis and anti-inflammatory evaluation of a series of novel 3-(indol-5-yl)-indazole derivatives with or without hydroxyethyl group. Among the tested compounds, compound 22m showed the highest anti-inflammatory activity in macrophages. Further, we explored the potential mechanism and molecular target of 22m through a series of biochemical assays. Interestingly, our data indicated that 22m directly bound to MD2-TLR4 complex and disrupted MD2-TLR4 activation. 22m also showed potent therapeutic effects in LPS-induced model of ALI. Our results provided a new lead compound, 22m, as potent MD2-TLR4 inhibitor for the treatment of ALI.


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Figure 2. Structures of 9-14 and drug design conception.

RESULTS AND DISCUSSION The syntheses of 3-(indol-5-yl)-indazole derivatives (22a-22q) reported in this study was performed as outlined in Schemes 1 and 2. Iodination of the commercially available 5-nitroindazole (15) with iodine in basic condition produced the 3-iodo-5-nitro-1H-indazole (16). The subsequent nucleophilic substitutions placed 2-(trimethylsilyl) ethoxymethyl (SEM) or benzyl substituent at N-1 on the indazole skeleton of 16, which were easily performed under basic conditions due to its relatively acidic character, thus affording N-1 substituted products 17a-17b. Furthermore, Suzuki coupling of 17a and 17b with (1H-indol-5-yl) boronic



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acid gave the key intermediates 18a and 18b,37 respectively.

Scheme 1. General synthetic route of 3-(indol-5-yl)-indazole derivatives 22a-22l. aReagents and conditions: (a) KOH, I2, DMF, rt; (b) NaH, SEMCl, DMF, 0 oC to rt; or BnCl, K2CO3, acetonitrile, reflux; (c) (1H-indol-5-yl)boronic acid, Na2CO3, PdCl(dppf), THF, reflux; (d) NaH, (2-bromoethoxy)-tert-butyldimethylsilane, DMF, 0

oC

to rt; (e) Fe, NH4Cl,

C2H5OH/H2O, reflux; (f) Substituted benzoyl chloride or acetyl chloride, DIPEA, CH2Cl2, 0


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oC

to rt ; (g) TBAF, EDA, THF, reflux.

N-alkylation

of

18a

with

(2-bromoethoxy)-tert-butyldimethylsilane

afforded

t-butyldimethylsilyl (TBS) protected product 19, which was reduced by catalytic iron and ammonia chloride in ethanol (EtOH), yielding the corresponding 6-aminoindazole derivative 20. Condensation of 20 with appropriate aliphatic or aromatic chloride (21a-21l), followed by removing of SEM and TBS groups, afforded target compounds 22a-22l.

Scheme 2. General synthetic route of 3-(indol-5-yl)-indazole derivatives 22m, 22n, 22o and 22p. aReagents and conditions: (a) Fe, NH4Cl, C2H5OH/H2O, reflux; (b) 2,6-dichlorobenzoyl



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chloride, DIPEA, CH2Cl2, 0 oC to rt; (c) TBAF, EDA, THF, reflux.

For the syntheses of compound 22m, key intermediate 18a was directly reduced to amine 23 by using activated iron power as a reducing agent, coupling with 2,6-dichlorobenzoic acid and then deprotection of SEM gave 22m (Scheme 2). 22n was also achieved with the same synthetic procedure. Alternatively, hydrolysis of the SEM and TBS groups of intermediates 19 and 20 yielding target compounds 22o and 22p, respectively. All title compounds (22a-22p) were fully characterized by proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic resonance (13C NMR), and electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The purity of chemicals was identified by HPLC method. LPS activates MD2-TLR4 on cell membranes and transduces signal to produce pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6.38, 39 Clinical evidence has shown that pro-inflammatory cytokines play a significant role in the pathogenesis of inflammatory-induced sepsis and ALI.40,

41

Hence, we established an enzyme-linked

immunosorbent assay (ELISA)42 to screen for the inhibitory activity of synthesized compounds (22a-22p) against LPS-induced TNF-α and IL-6 release in mouse primary macrophages

(MPMs).

Previously

reported

anti-inflammatory

compound

5-(4-methylpiperazin-1-yl)-3-propyl-2-(1H-pyrrolo[2,3-b]pyridin-3-yl)-3H-imidazo[4,5-b]-py ridine (X12)43 was used as positive control, and DMSO was used as the vehicle control. The cytokine inhibitory activity of the compounds is summarized in Figure 3. Our screening results show that majority of the compounds tested exhibit greater inhibition of LPS-induced TNF-α and IL-6 release compared to X12. We also evaluated their cytotoxicity in MPMs


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using the MTT method. As shown in Supplementary Figure S1A, all compounds at 10 μM showed no cytotoxicity. Among these compounds, 22c, 22d, 22g, 22m, 22n and 22o inhibited IL-6 release with rates greater than 50%. Meanwhile, 22c, 22g, 22m, 22o and 22p showed high inhibitory rates against TNF-α release. Notably, 22m inhibited both TNF-α and IL-6 release with inhibitory rates reaching 83% and 91%, respectively, compared to the LPS control.

Figure 3. Effect of 3-(indol-5-yl)-indazole derivatives on LPS-induced cytokine production in macrophages. The effect of compounds on LPS-induced TNF-α (A) and IL-6 (B) release in



mouse primary peritoneal macrophages (MPMs) was measured by ELISA. Cells were pretreated with vehicle, X12 and compounds 22a-22p (10 μM) for 2 h. Cells were then treated with LPS (0.5 μg/mL) for 22 h. Each bar represents mean ± SEM of 3 independent experiments. Statistical significance relative to LPS group is indicated (*p < 0.05, **p < 0.01, ***p < 0.001 compared to LPS).

To our knowledge, there are few studies that have reported on the cytokine-inhibitory effects of 3-(indol-5-yl)-indazole derivatives and their structure-activity relationship (SAR) analysis. Based on the results shown in Figure 3, compound 22o with electron-withdrawing group (-NO2) at the 5-position of indazol core shows relatively potent inhibitory effect against TNF-α and IL-6 compared to X12. However, after the reduction to amine group (22p), the inhibitory activity against IL-6 dramatically declines. Next, we investigated the importance of the substitutions at the 5-position of indazol core. After modification of acetamide group, resulting compound 22a showed increased inhibitory activity against IL-6 but not TNF-α. When the acetamide group was replaced with a propanamide group (22b), the potencies against both IL-6 and TNF-α were almost completely abolished. However, further investigation revealed that the potency loss of compound 22b was partially restored by introducing a 3-chloropropanamide (22c) at the 5-position of indazol core. We next examined substituted N-aryl formamide moieties by synthesizing 22d-22k. Incorporation of a chlorine atom at the meta-position of the phenyl ring provided 22d, which displayed significant increases in potency against IL-6 release. Moreover, introduction of a chlorine (22e) or fluoro (22f) atom at para-position of the phenyl ring obviously decreased the


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anti-inflammatory potency. When 2,6-dichlorobenzamide was introduced (22g), the ability against TNF-α remarkably increased compared to 22d with an approximately 47% inhibitory rate. Comparatively, modification of electron-donating groups like 3-methyl (22h), 3,5-dimethoxy group (22i) or heterocycles (22j and 22k), led to a dramatic loss in inhibitory activity against both IL-6 and TNF-α. Interestingly, the introduction of cinnamamide moiety yielding 22l, which contains a Michael receptor, exhibited very weak anti-inflammatory activity. These results implied that halogen group substituted benzamide was crucial for the anti-inflammatory effect. Compared to 22g, the removal of ethanol group at R3 position yielding 22m was found to dramatically improve the activities against TNF-α and IL-6 (83% and 91% inhibitory rates, respectively), indicating that the hydroxyethyl group may not contribute to the anti-inflammatory benefits. Further introduction of a bulky group like benzyl into indazole core of 22m afforded 22n, which demonstrated decreased anti-inflammatory potency. Taken together, the preliminary SAR analysis for the TNF-α and IL-6 inhibitory activities of the novel 3-(indol-5-yl)-indazole derivatives may provide valuable information for the further development of anti-inflammatory compounds. Among the screened compounds, 22c, 22m and 22o showed most promising effects against IL-6 and TNF-α release. Thus, we selected these compounds for further investigation of potential dose-dependent cytokine inhibitory effects. We also included compound 22g, which significantly inhibited IL-6 and appeared to show inhibitory effect on TNF-α as well. MPMs were pretreated with the three active compounds in increasing concentrations (0.5, 1, 2.5, 5.0, 10, and 20 μM) for 30 min. Cells were then challenged with 0.5 μg/mL LPS for 24 h. The release of TNF-α and IL-6 in the culture medium was determined by ELISA. As shown



in Figure 4, all four active compounds exhibited a dose-dependent inhibition of LPS-induced cytokines. In particular, compounds 22g, 22m and 22o suppressed LPS-induced IL-6 with IC50 values in the 0.5-1.0 micromole range (IC50 values = 0.73 μM, 0.53 μM and 0.84 μM, respectively). Interestingly, 22m showed suppressed TNF-α release with an IC50 value of 0.89 μM, which is obviously smaller compared to values obtained with compounds 22c, 22g and 22o (3.47, 6.31 and 6.99 μM). As expected, 22m did not show cytotoxicity in MPMs in the concentration range from 0.5 to 20 μM (Supplementary Figure S1B). These results suggest that 22m may be a promising anti-inflammatory candidate for the treatment of ALI and sepsis.


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Figure 4. 3-(indol-5-yl)-indazole derivatives inhibited LPS-induced inflammatory cytokines in a dose-dependent manner. MPMs were pretreated with vehicle or compounds at increasing concentrations for 2 h. Subsequently, cells were incubated with LPS (0.5 μg/mL) for 22 h. (A) IL-6 and (B) TNF-α levels as measured by ELISA. Each bar represents mean ± SEM of 3 independent. Statistical significance relative to the LPS group is indicated (*p < 0.05, **p < 0.01 compared to LPS).

As mentioned earlier, upon LPS recognition, the MD2-TLR4 mediates cytokine gene expression through the activation of the nuclear factor (NF)-κB. Hence, we examined whether compound 22m altered LPS-induced NF-κB signaling. We prepared nuclear fractions from MPMs challenged with LPS. As shown in Figure 5A, the NF-κB p65 subunit nuclear levels were increased in MPMs exposed to LPS. Pretreatment of cells with 22m suppressed the levels indicating inhibition of NF-κB activation. We confirmed these results by infecting RAW 264.7 macrophages with NFκB-EGFP reporter. As shown in Figure 5B and 5C, flow cytometric analysis indicated that 22m significantly reduced the transcriptional activity of NF-κB as detected by green fluorescent protein (GFP) fluorescence. These data show that 22m exerts its anti-inflammatory activity by inhibiting NF-κB.



Figure 5. Compound 22m attenuated LPS-induced activation of NF-κB signaling. (A) Western blot analysis of NF-κB p65 subunit levels in nuclear fractions. Macrophages were challenged with LPS, with or without pretreatment with compound 22m. Lamin B was used as loading control. (B, C) NF-κB reporter assay showing suppressed activation of NF-κB following 22m treatment. RAW264.7 cells stably expressing NF-κB-EGFP reporter were subjected to flow cytometry (B) to detect NF-κB activation. Quantification of fluorescence is shown in panel C (**p < 0.01 compared to Ctrl, ##p < 0.01 compared to LPS). (D) Schematic illustration showing MD2-TLR4 signaling initiated by LPS. (E) Kinase selectivity profile of compound 22m against 98 kinases.


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A schematic diagram showing mechanisms of LPS trafficking and TLR4-mediated inflammatory signaling is presented in Figure 5D. The activated receptor dimer of (LPS-MD-2-TLR4)2 recruits the intracellular adaptor MyD88 to activate NF-κB through a cascade of multiple kinase phosphorylation. The proteins involved between MyD88 and NF-κB are complex and include kinases such as IL-1 receptor-associated kinase 4 (IRAK4), IRAK1, transforming growth factor-β-activated kinase 1 (TAK1), inhibitory κB kinase α and β (IKKα, IKKβ), and mitogen-activated protein kinases (MAPK). To identify the possible molecular target of 22m in this cascade, we profiled the inhibitory activity of 22m against a panel of 98 kinases using DiscoverX’s KINOMEscan platform. This panel comprises majority of proteins invovled in the MyD88-NF-κB cascade as well as other proteins distributed across the whole kinome (Supplementary Table S1). Our results show that 22m did not inhibit any of these 98 kinases (Figure 5E and Supplementary Table S1). These results indicate that kinases in MyD88-NF-κB cascade may not be targeted by 22m.



Figure 6. Identification of MD2-TLR4 as the molecular target of compound 22m. (A) SPR analysis of 22m to binding to recombinant MD2 protein; (B) SPR analysis of 22m to binding to TLR4 protein; (C) SPR analysis of 22m to binding to MyD88 protein. (D-E) Co-immunoprecipitation showing effect of 22m pretreatment on MD2-TLR4 complex formation in macrophages exposed to LPS. Panel E showing quantification of immunoreactivity (*p < 0.05 compared to control; #p < 0.05 compared to LPS). (F) TLR4 dimerization in HEK293T co-expressing HA and Flag-tagged TLR4. (G) Flow cytometric analysis of the effect of 22m on binding of FITC-labeled LPS on macrophage cell surface. (H) Molecular docking analysis of 22m with MD2-TLR4 complex.

Based on our findings, we postulated that proteins upstream of MyD88-NF-κB cascade


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might be the target of 22m. To test this, we examined the direct interaction of 22m with recombinant MD2, TLR4, and MyD88 proteins by surface plasmon resonance (SPR) analysis. As shown in Figure 6A-C, 22m binds to recombinant MD2 and TLR4 protein with a KD value of 185 and 146 μM, respectively, while it did not interact with intracellular adaptor protein MyD88. These data suggest that MD2-TLR4 may be the direct target of 22m in producing its anti-inflammatory activity. To confirm MD2-TLR4 as the target of 22m, we performed co-immunoprecipitation to investigate MD2-TLR4 complex formation. Our results show that 22m significantly inhibited dimerization of the MD2-TLR4 complex in LPS-activated MPMs (Figure 6D-E). We then constructed Flag-TLR4 and HA-TLR4 plasmids to investigate the dimerization of TLR4. In Flag-TLR4 and HA-TLR4 co-expressing HEK293 cells, 22m inhibited the homodimerization of TLR4 as determined by co-immunoprecipitation analysis (Figure 6F). We then investigated the ability of 22m to compete with LPS binding to MD2-TLR4 complex at the cellular level. MPMs were challenged with fluorescein isothiocyanate (FITC) -labeled LPS (FITC-LPS) with or without 22m pretreatment and fluorescence was analyzed by flow cytometry. Here we show that pretreatment with 22m significantly inhibited FITC-LPS binding to the cell surface (Figure 6G). These data validate the interaction of 22m with its molecular target MD2/TLR4. By the way, although we excluded the kinases using kinome assay, we do not know if 22m can target other TLRs. There are 10 human TLR members in the TLR family. This is a limitation of this study. The measurement of the interactions between 22m and recombinant proteins of other TLRs to clarify the specificity of 22m to TLR4 will be needed in the future. We then performed molecular docking analysis to investigate the binding mode between



22m and MD2-TLR4 complex (PDB: 3FXI44). In our simulations, the 2,6-dichlorobenzamide part of 22m inserted deep into the MD2 hydrophobic pocket and interacted with the inside hydrophobic residues such as Ile-52, Phe-121, Phe-126 and Ile-153 (Figure 6H). The indole fragment of 22m stretches to the rim of MD2 pocket and formed a key hydrogen bond with Lys-125 residue of MD2. The indazole group locates to the MD2-TLR4 interface, further forming a hydrogen bond with Glu-439 residue of TLR4. Excessive production of inflammatory cytokines induced by LPS leads to coagulation, endothelial damage and microvascular leakage, resulting in lung organ dysfunction and injury.45, 46 We, therefore, investigated the potential effect of 22m on ALI in LPS-challenged mice. Histological analysis showed significant lung edema, pulmonary congestion, neutrophilic infiltration and thickening of the alveolar wall in lung tissues of mice challenged with LPS (Figure 7A). Pretreatment of mice with compound 22m effectively reduced airspace inflammation and amended the tissue structure of pulmonary lobules. Scoring of lung injury showed that 22m significantly reduces injury score to levels comparable to X12 (Figure 7B). These results indicate that 22m is protective against LPS-induced ALI.


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Figure 7. Effect of 22m on histopathological changes in lung tissues of mice challenged with LPS. (A) H&E staining of lung sections. (B) Lung injury scores (n=8 per group; ns: no significance; *p < 0.05, **p < 0.01 compared to LPS).

Myeloperoxidase (MPO) is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies, and serves as a marker of neutrophil accumulation within tissues.47, 48 To examine the effect of 22m on neutrophil lung infiltration, we measured MPO activity in lung tissues harvested from LPS-challenged mice. Figure 8A shows that the treatment of mice with 22m at 5 and 10 mg/kg significantly decreases LPS-induced MPO activity. Under the same experimental conditions, 22m reduced LPS-induced serum TNF-α levels (Figure 8B).



Figure 8. 22m attenuate LPS-induced lung inflammation and pulmonary macrophage infiltration in mice. Male C57BL/6 mice were treated with compounds by tail vein injection 15 min before an intraperitoneal injection of LPS (20 mg/kg). Mice were anesthetized and sacrificed 6 h after LPS injection. Serum and lung tissues were collected. (A) MPO activity in lung tissue lysates (**p < 0.01 compared to LPS). (B) Serum levels of TNF-α (**p < 0.01 compared to LPS). (C) F4/80 immunohistochemical staining of lung tissues showing increased macrophage infiltration following LPS challenge. Compound 22m prevented macrophage infiltration as seen by F4/80 staining (brown). (D) Quantification of F4/80-positive cells (**p < 0.01 compared to LPS).

To investigate whether 22m prevented macrophage infiltration in the LPS-induced ALI


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model, we utilized F4/80, a widely used marker of macrophages.49 Immunohistochemical analysis revealed increased F4/80 immunoreactivity in lung tissues harvested from LPS-challenged mice, while F4/80 positive macrophages decreased significantly with 22m and X12 treatment (Figure 8C and 8D). This indicates that 22m prevented macrophage infiltration following LPS challenge. We next investigated the inhibitory effect of compound 22m on the mRNA levels of a panel of proinflammatory factors in lung tissues. Real-time qPCR analysis revealed that treatment of LPS-challenged mice with 22m decreased the mRNA levels of TNF-α, IL-6, IL-1β, IL-12 and IL-33 in lung tissues (Figure 9). In addition, cyclooxygenase-2 (COX-2) mRNA levels were also reduced upon 22m treatment. These results suggest that 22m can inhibit macrophage infiltration and reduce proinflammatory cytokine production in lung tissues of ALI mice.

Figure 9. 22m reduced mRNA levels of inflammatory genes induced by LPS in mouse lung tissues. (A-F) mRNA levels of (A) TNF-α, (B) IL-6, (C) IL-1β, (D) COX-2, (E) IL-12, and (F) IL-33 as assessed by quantitative RT-PCR (data normalized to β-actin; n=8 per group; *p



< 0.05, **p < 0.01 compared to LPS).

CONCLUSIONS Acute inflammation is the leading cause of ALI, and the rate of hospitalization of patients with ALI is increasing.50 Thus, there is a pressing need to identify new anti-inflammatory molecules and drug targets to prevent or mitigate inflammatory responses in these patients. Numerous studies demonstrated that inhibiting MD2-TLR4 complex suppresses LPS-induced inflammatory responses and the development of ALI in experimental models. Therefore, antagonists directly targeting MD2-TLR4 have emerged as attractive pharmacological targets for ALI and other inflammatory disorders.51 In the present study, we have designed and synthesized a novel series of 3-(indol-5-yl)-indazole derivatives and evaluated their anti-inflammatory activity. Active compound 22m inhibited LPS-induced TNF-α and IL-6 production in macrophages. We also showed that 22m leads to reduced activity of NF-κB. However, kinase phosphorylation profiling showed that 22m may not directly inhibit kinases involved in the MD2-TLR4 pathway. Then, we found that 22m directly bound MD2 and TLR4 complex through a series of molecular studies including SPR, co-immunoprecipitation assays, flow cytometric determination of LPS binding, and molecular docking. In vivo investigation in LPS-challenged mice showed that treatment with 22m significantly decreases macrophage infiltration, MPO activity, and inflammatory cytokines. The outcome of 22m treatment was normalization of pulmonary histopathological changes. Although the Kd values of 22m-MD2 and 22m-TLR4 interactions are about 150μM, higher than that of the previously reported


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MD2 inhibitor 2i (Kd=34μM)

52,

22m represents a new lead structure targeting MD2/TLR4

complex. Taken together, our studies show that 3-(indol-5-yl)-indazole derivative 22m is a new lead for the design and discovery of MD2-TLR4 antagonists for the treatment of ALI and potentially other inflammatory diseases.

EXPERIMENTAL SECTION General Synthetic Chemistry Methods.All reagents and solvents were purchased from Alfa Aesar and Sigma-Aldrich, respectively. Other chemicals were obtained from local suppliers and were used without further purification. All reactions were monitored by thin-layer chromatography (250 silica gel 60 F254 glass plates). Melting points were determined on a Fisher-Johns melting apparatus and were uncorrected. Flash chromatography was performed using Silica Gel 60 (E. Merck, 70-230 mesh). 1H NMR spectra was recorded on a Bruker 400, 500 and 600 MHz instruments, and the chemical shifts were expressed in terms of parts per million with TMS as the internal reference. High-resolution mass spectrometry (HRMS) was measured with an Agilent G6520 quadrupole time-of-flight mass spectrometer in electrospray mode. Analytical HPLC analyses were performed at ambient temperature on Agilent 1260 liquid chromatograph fitted with Inertex C18 column (4.6 mm × 150 mm, 5 μm particle size). All general chemicals were the highest available grade. The purity of all synthetic compounds was determined by HPLC analysis and was higher than 95%.

3-Iodo-5-nitro-1H-indazole (16). I2 (15.66 g, 61.6 mmol) and KOH pellets (3.58 g, 63.7 mmol) were successively added to a stirred solution of 5-nitro-1H-indazole (15, 5.0 g, 30.8



o

mmol) in DMF (60 mL) at 65 C. After 3 h, the result mixture was poured into 1M HCl (60 mL) and 10% Na2S2O3 (100 mL). The mixture was stirred for 20 min, and the precipitate was collected. The solid was dissolved in EtOAc, and the insoluble residue was filtered. The filtrate was evaporated to give compound 16 (7.99 g, 90%) as a yellow solid. m.p 157.5-160.6 oC. 1H

NMR (500 MHz, CD3OD) δ (ppm) 8.42 (s, 1H, H-4), 8.30 (d, J = 7.5 Hz, 1H, H-7),

7.66 (d, J = 5.0 Hz, 1H, H-6). 13C NMR (125 MHz, CD3OD) δ (ppm) 144.26, 144.18, 128.18, 123.44, 119.59, 112.35, 96.39. ESI-MS m/z: 287.6 (M-H)-, calcd for C7H4IN3O2: 288.93.

3-Iodo-5-nitro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-indazole (17a). To a solution of compound 16 (2.0 g, 6.92 mmol) and NaH (80%, 540 mg, 17.99 mmol) in DMF (20 mL) was added 2-(trimethylsilyl)ethoxymethyl chloride (1.50 mL, 8.30 mmol) in an ice bath. Subsequently, the reaction mixture was stirred at room temperature for 2.5 h and poured into ice water (50 mL). The mixture was stirred for 30 min, and the precipitate was collected. The residue was purified by flash chromatography to give compound 17a (2.6 g, 90%) as a yellow solid. m.p 87.7-88.4 oC. 1H NMR (500 MHz, CDCl3) δ (ppm) 8.72-8.27 (m, 2H, H-6, H-7), 7.69 (s, 1H, H-4), 5.78 (s, 2H, NCH2O), 3.62 (s , 2H, OCH2CH2Si(CH3)3), 0.92 (s, 2H, OCH2CH2Si(CH3)3), -0.02 (s, 9H, OCH2CH2Si(CH3)3). 13C NMR (125 MHz, CDCl3) δ (ppm) 143.47 (C-8), 142.33 (C-5), 128.80 (C-9), 122.88 (C-6), 119.43 (C-4), 110.59 (C-7), 95.34 (C-3), 78.57 (NCH2O), 67.25 (OCH2CH2Si(CH3)3), 17.72 (OCH2CH2Si(CH3)3), -1.48 × 3 (Si(CH3)3). ESI-MS m/z: 420.9 (M+Na)+, calcd for C13H18IN3O3Si: 419.2.

1-Benzyl-3-iodo-5-nitro-1H-indazole (17b). A mixture of 16 (0.4 g, 1.38 mmol), K2CO3


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(0.38 g, 2.76 mmol) and benzyl bromide (164 μL, 1.38 mmol) in acetonitrile (20 mL) was heated to reflux for 1 h. The acetonitrile was removed in vacuo, and the resulting residue was extracted with EtOAc for three times. The organic layer was separated and dried over anhydrous MgSO4, concentrated, and purified by flash chromatography to afford compound 17b (0.48 g, 95%) as a yellow solid. m.p 151.5-153.2 oC. 1H NMR (500 MHz, CDCl3) δ (ppm) 8.48 (s, 1H, H-4), 8.25 (d, J = 10.0 Hz, 1H, H-7), 7.37-7.23 (m, 6H, H-6, Bn-(H2-H6)), 5.64 (s, 2H, Bn-CH2).

13C

NMR (125 MHz, CDCl3) δ (ppm) 143.00 (C-5), 142.10 (C-8),

135.18 (Bn-C1), 129.06 (Bn-C3, Bn-C5), 128.50 (Bn-C4), 128.42 (C-9), 127.34 (Bn-C2, Bn-C6), 122.56 (C-6), 119.62 (C-4), 110.07 (C-7), 94.44 (C-3), 54.26 (Bn-CH2). ESI-MS m/z: 380.1 (M+H)+, calcd for C14H10IN3O2: 379.8.

3-(1H-Indol-5-yl)-5-nitro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-indazole

(18a).

A

mixture of compound 17a (3.48 g, 8.30 mmol), aqueous 2 M Na2CO3 (33 mL, 66 mmol), PdCl2(dppf)·DCM (1.63 g, 2 mmol), and 5-indolylboronic acid (1.77 g, 9.96 mmol) in THF (33 mL) was heated to reflux for 5 h under an atmosphere of nitrogen. The mixture was allowed to cool to room temperature and diluted with H2O and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography to give compound 18a (2.74 g, 67%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ (ppm) 9.06 (s,1H, NH), 8.42 (s, 1H, H-4), 8.34 (d, J = 12.0 Hz, 1H, H-7), 8.22 (s, 1H, H-4'), 7.79 (d, J = 8.0 Hz, 1H, H-6'), 7.66 (d, J = 12.0 Hz, 1H, H-6), 7.57 (d, J = 8.0 Hz, 1H, H-7'), 7.31 (s, 1H, H-2'), 6.70 (s, 1H, H-3'), 5.83 (s, 2H, N-CH2-O), 3.67 (t, J = 8.0 Hz, 2H, OCH2CH2Si(CH3)3), 0.93 (t, J = 8.0 Hz, 2H,



Page 28 of 63

OCH2CH2Si(CH3)3), -0.05 (s, 9H, OCH2CH2Si(CH3)3). 13C NMR (125 MHz, CDCl3) δ (ppm) 148.94 (C-3), 143.15 (C-8), 142.93 (C-5'), 136.24 (C-5), 128.37 (C-8'), 123.46 (C-9'), 122.26 (C-9), 121.96 (C-6), 120.41 (C-4), 119.86 (C-4'), 111.92 (C-6'), 110.23 (C-7, C-7'), 103.36 (C-3'), 67.02 (OCH2CH2Si(CH3)3), 17.79 (OCH2CH2Si(CH3)3), -1.41 (OCH2CH2Si(CH3)3). ESI-MS m/z: 409.1 (M+H)+, calcd for C21H24N4O3Si: 408.6.

1-Benzyl-3-(1H-indol-5-yl)-5-nitro-1H-indazole (18b). Following general procedure for the synthesis of 17a, the crude residue was purified by flash chromatography to furnish compound 18b (177 mg, 61%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ (ppm) 9.07 (s, 1H, NH), 8.41 (s, 1H, H-4), 8.31-8.19 (m, 1H, H-7), 7.82 (d, J = 8.0 Hz, 1H, H-6), 7.56 (d, J = 8.4 Hz, 1H, H-4'), 7.46-7.24 (m, 7H, Bn-(H2-H6), H2', H7'), 6.70 (s, 1H, H-3'), 5.70 (s, 2H, Bn-CH2). 13C NMR (125 MHz, DMSO-d6) δ (ppm) 148.76 (C-3), 142.78 (C-8, C-5'), 142.51 (C-5), 136.24 (Bn-C1), 135.94 (C-8'), 128.94 (Bn-C3, Bn-C5), 128.44 (Bn-C2, Bn-C6), 128.17 (Bn-C4), 127.25 (C-2'), 125.26 (C-9), 123.74 (C-6), 121.99(C-4), 121.68 (C-4'), 121.58 (C-6'), 120.28 (C-7'), 120.01 (C-7), 103.39 (C-3'), 53.54 (Bn-CH2). ESI-MS m/z: 368.7 (M+H)+, calcd for C22H16N4O2: 367.1.

3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-5-nitro-1-{[2-(trimethylsilyl) ethoxy]methyl}-1H-indazol (19) . To a mixture of compound 18a (1.0 g, 2.44 mmol) and NaH

(190

mg,

6.34

mmol)

in

DMF

(9

mL)

was

added

(2-bromoethoxy)(tert-butyl)dimethylsilane (703 mg, 2.94 mmol) in an ice-bath. Subsequently, the reaction mixture was stirred at room temperature for 3 h. The resulting solution was


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quenched with redistilled water and extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered, and evaporated. The residue was purified by flash chromatography (silica gel; Hexane/EtOAc, gradient, 10-30% EtOAc) to afford compound 19 (1.11 g, 86%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ (ppm) 9.06 (s, 1H, H-4), 8.34 (d, J = 10.0 Hz, 1H, H-7), 8.19 (s, 1H, H-4'), 7.79 (d, J = 10.0 Hz, 1H, H-6), 7.66 (d, J = 10.0 Hz, 1H, H-6'), 7.51 (d, J = 10.0 Hz, 1H, H-7'), 5.83 (s, 2H, N-CH2-O), 4.30 (t, J = 8.0 Hz, 2H, OCH2CH2Si(CH3)3), 3.97 (t, J = 8.0 Hz, 2H, N-CH2CH2-O), 3.67 (t, J = 8.0 Hz, 2H, N-CH2CH2-O), 0.93 (t, J = 8.0 Hz, 2H, OCH2CH2Si(CH3)3), 0.84 (s, 9H, O-Si(CH3)2C(CH3)3), -0.05 (s, 9H, OCH2CH2Si(CH3)3), -0.11 (s, 6H, O-Si(CH3)2C(CH3)3). 13C

NMR (125 MHz, CDCl3) δ (ppm) 149.04 (C-3), 143.00 (C-5', C-8), 129.77 (C-5), 129.14

(C-8'), 121.84 (C-9'), 121.31 (C-6, C-9), 120.54 (C-4), 119.86 (C-4'), 110.17 (C-6'), 110.13 (C-7, C-7'), 101.93 (C-3'), 78.21 (N-CH2-O), 66.98 (OCH2CH2Si(CH3)3), 62.43 (N-CH2CH2-O),

48.98

(N-CH2CH2-O),

25.81

(O-Si-(CH2)2C(CH3)3,

25.65

(O-Si-(CH2)2C(CH3)3), 17.81 (OCH2CH2Si(CH3)3), -1.45 (OCH2CH2Si(CH3)3), -5.66×2 (O-Si-(CH2)2C(CH3)3). ESI-MS m/z: 567.2 (M+H)+, calcd for C29H42N4O4Si2: 566.3.

3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-1-{[2-(trimethylsilyl)ethoxy]m ethyl}-1H-indazol-5-amine (20) . The compound 19 (1.15 g, 2.03 mmol) was dissolved in ethanol (40 mL) and water (10 mL). Iron powder (0.98 g, 17.44 mmol) and ammonium o

chloride (0.24 g, 4.46 mmol) were then added, and the resulting mixture was heated to 78 C for 4 h. The ethanol was removed in vacuo, and the resulting residue was basified with sodium bicarbonate and extracted with EtOAc. The organic layer was separated and dried



Page 30 of 63

over anhydrous MgSO4, filtered and evaporated. The residue was purified by chromatography to afford compound 20 (0.97 g, 91%) with a brown oil. 1H NMR (400 MHz, CDCl3) δ (ppm) 8.13 (s, 1H, H-6'), 7.77 (d, J = 12.0 Hz, 1H, H-7), 7.44 (t, J = 8.0 Hz, 2H, H-7'), 7.34 (s, 1H, H-4'), 7.19 (s, 1H, H-4), 6.94 (d, J = 8.0 Hz, 1H, H-6), 6.56 (s, 1H, NH), 5.74 (s, 2H, N-CH2-O), 4.27 (t, J = 5.6 Hz, 2H, OCH2CH2Si(CH3)3), 3.95 (t, J = 5.6 Hz, 2H, N-CH2CH2-O), 3.63 (t, J = 8.0 Hz, 2H, N-CH2CH2-O), 0.91 (t, J = 8.0 Hz, 2H, OCH2CH2Si(CH3)3), 0.84 (s, 9H, O-Si(CH3)2C(CH3)3), -0.06 (s, 9H, OCH2CH2Si(CH3)3), -0.12 (s, 6H, O-Si(CH3)2C(CH3)3).

13C

NMR (100 MHz, CDCl3) δ (ppm) 144.92 (C-3),

140.50 (C-5, C-5'), 136.77 (C-8'), 135.86 (C-9'), 128.86 (C-8), 124.87 (C-2'), 123.66 (C-9), 121.39 (C-4'), 119.97 (C-6), 118.40 (C-6'), 110.54 (C-7'), 109.54 (C-7), 104.80 (C-4), 101.46 (C-3'), 77.70 (N-CH2-O), 66.17 (OCH2CH2Si(CH3)3), 62.36 (N-CH2CH2-O), 48.82 (N-CH2CH2-O),

25.78

(O-Si-(CH2)2C(CH3)3),

18.18

(O-Si-(CH2)2C(CH3)3),

17.75

(OCH2CH2Si(CH3)3), -1.52 (OCH2CH2Si(CH3)3), -5.72 (O-Si-(CH2)2C(CH3)3). ESI-MS m/z: 537.5 (M+H)+, calcd for C29H44N4O2Si2: 536.3.

General Procedure for Synthesis of 21a-l. To a solution of compound 20 (0.14 g, 0.26 mol) in anhydrous DCM (6 mL) was added N,N-Diisopropylethylamine (0.07 mL, 0.39 mmol) and various acyl chloride (0.52 mmol) in an ice-bath. The reaction mixture was stirred at room temperature and monitored by TLC. After completion of the reaction, the resulting mixture was quenched with saturated aqueous ammonium chloride and extracted with EtOAc. The organic layer was separated and dried over anhydrous MgSO4, filtered and evaporated. The residue was purified by flash chromatography to afford the compounds 21a-l (71-83%).


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N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-1-{[2-(trimethylsilyl) ethoxymethyl]-1H-indazol-5-yl}acetamide (21a). Yellow oil. 78.5% yield. 1H NMR (400 MHz, CDCl3) δ (ppm) 8.22 (s, 1H, H-4), 8.15 (s, 1H, H-4'), 7.78 (d, J = 8.5 Hz, 1H, H-7), 7.56 (s, 1H, H-6'), 7.51 (s, 1H, H-7'), 7.43 (d, J = 8.5 Hz, 1H, H-6), 7.18 (d, J = 3.0 Hz, 1H, H-2'), 6.55 (d, J = 3.0 Hz, 1H, H-3'), 5.76 (s, 2H, N-CH2-O), 4.26 (t, J = 5.5 Hz, 2H, OCH2CH2Si(CH3)3), 3.93 (t, J = 5.5 Hz, 2H, N-CH2CH2-O), 3.62 (t, J = 8.0 Hz, 2H, N-CH2CH2-O), 2.19 (s, 3H, NCOCH3), 0.90 (t, J = 8.0 Hz, 2H, OCH2CH2Si(CH3)3), 0.84 (s, 9H,

O-Si(CH3)2C(CH3)3),

-0.07

(s,

9H,

OCH2CH2Si(CH3)3),

-0.13

(s,

6H,

O-Si(CH3)2C(CH3)3). ESI-MS m/z: 579.42 (M+H)+, calcd for C29H44N4O2Si2: 578.31.

N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl)-1H-indol-5-yl}-1-{[2-(trimethylsilyl)ethox y]methyl}-1H-indazol-5-yl}propionamide (21b). Colorless oil. 50.4% yield. 1H NMR (500 MHz, CDCl3) δ 8.26 (s, 1H, H-4), 8.15 (s, 1H, H-4'), 7.78 (d, J = 8.5 Hz, 1H, H-7), 7.52 (s, 1H, H-7'), 7.43 (d, J = 8.5 Hz, 1H, H-6), 7.18 (s, 1H, H-2'), 6.56 (s, 1H, H-3'), 5.77 (s, 2H, N-CH2-O), 4.26 (t, J = 5.5 Hz, 2H, OCH2CH2Si(CH3)3), 3.94 (t, J = 5.5 Hz, 2H, N-CH2CH2-O), 3.62 (t, J = 8.0 Hz, 2H, N-CH2CH2-O), 2.42 (q, J = 6.5 Hz, 2H, NCOCH2CH3 ), 1.27 (t, J = 6.3 Hz, 3H, NCOCH2CH3), 0.92 (t, J = 8.5 Hz, 2H, OCH2CH2Si(CH3)3), 0.84 (s, 9H, O-Si(CH3)2C(CH3)3), -0.06 (s, 9H, OCH2CH2Si(CH3)3), -0.12 (s, 6H, O-Si(CH3)2C(CH3)3).

N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-1-{[2-(trimethylsilyl)ethox y]methyl}-1H-indazol-5-yl}-3-chloropropanamide (21c). Colorless oil. 45.5% yield. 1H



NMR (500 MHz, CDCl3) δ 8.24 (s, 1H, H-4), 8.14 (s, 1H, H-4'), 7.78 (d, J = 7.9 Hz, 1H, H-7), 7.54 (m, 3H, NH, H-6, H-7'), 7.44 (d, J = 8.5 Hz, 1H, H-6'), 7.19 (d, J = 2.8 Hz, 1H, H-2'), 6.56 (d, J = 2.5 Hz, 1H, H-3'), 5.77 (s, 2H, N-CH2-O), 4.27 (t, J = 5.0 Hz, 2H, OCH2CH2Si(CH3)3), 3.94 (t, J = 5.5 Hz, 2H, N-CH2CH2-O), 3.90 (t, J = 6.5 Hz, 2H, Cl-CH2CH2-), 3.62 (t, J = 8.0 Hz, 2H, N-CH2CH2-O), 2.84 (t, J = 6.0 Hz, 2H, Cl-CH2CH2-), 0.91 (t, J = 8.0 Hz, 2H, OCH2CH2Si(CH3)3), 0.84 (s, 9H, O-Si(CH3)2C(CH3)3), -0.06 (s, 9H, OCH2CH2Si(CH3)3), -0.12 (s, 6H, O-Si(CH3)2C(CH3)3).

N-{3-{1-{2-[(tert-butyldimethylsilyl)oxy]ethyl}-1H-indol-6-yl}-1-{[2-(trimethylsilyl)ethox y]methyl}-1H-indazol-5-yl}-3-chlorobenzamide (21d). Yellow powder. 82.3% yield. 1H NMR (400 MHz, CD3OD) δ (ppm) 8.52 (s, 1H, H-4), 8.12 (s, 1H, H-4'), 7.99 (s, 1H, H-2''), 7.89 (d, J = 7.8 Hz, 1H, H-7), 7.74 (d, J = 8.7 Hz, 2H, H-6', H-6), 7.65 (d, J = 9.0 Hz, 1H, H-7'), 7.61-7.44 (m, 3H, H-4'', H-5'', H-6''), 7.27 (d, J = 3.0 Hz, 1H, H-2'), 6.54 (d, J = 3.0 Hz, 1H, H-3'), 5.78 (s, 2H, N-CH2-O), 4.30 (t, J = 4.8 Hz, 2H, OCH2CH2Si(CH3)3), 3.96 (t, J = 4.8 Hz, 2H, N-CH2CH2-O), 3.65 (t, J = 8.0 Hz, 2H, N-CH2CH2-O), 0.88 (t, J = 7.6 Hz, 2H, OCH2CH2Si(CH3)3), 0.79 (s, 9H, O-Si(CH3)2C(CH3)3), -0.07 (s, 9H, OCH2CH2Si(CH3)3), -0.18 (s, 6H, O-Si(CH3)2C(CH3)3). ESI-MS m/z: 675.39 (M+H)+, calcd for C36H47ClN4O3Si2: 674.28.

N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-1-{[2-(trimethylsilyl)ethox y]methyl}-1H-indazol-5-yl}-4-chlorobenzamide (21e) . Colorless oil. 78.2% yield. 1H NMR (500 MHz, CDCl3) δ 8.31 (s, 1H, H-4), 8.19 (s, 1H, H-4'), 8.17 (s, 1H, NH), 7.80 (d, J = 7.5


Page 32 of 63

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Hz, 3H, H-7, H-2'', H-6''), 7.60 (d, J = 7.3 Hz, 1H, H-6'), 7.50 (d, J = 8.7 Hz, 1H, H-6), 7.36-7.42 (m, 3H, H-7', H-3'', H-5''), 7.17 (d, J = 2.8 Hz, 1H, H-2'), 6.54 (d, J = 2.4 Hz, 1H, H-3'), 5.75 (s, 2H, N-CH2-O), 4.24 (s, 2H, OCH2CH2Si(CH3)3), 3.93 (s, 2H, N-CH2CH2-O), 3.75 – 3.55 (m, 2H, N-CH2CH2-O), 0.99 – 0.77 (m, 11H, OCH2CH2Si(CH3)3, O-Si(CH3)2C(CH3)3), 0.06--0.18 (m, 15H, OCH2CH2Si(CH3)3, O-Si(CH3)2C(CH3)3).

N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-1-{[2-(trimethylsilyl)ethox y]methyl}-1H-indazol-5-yl}-4-fluorobenzamide (21f). Colorless oil. 79.5% yield. 1H NMR (500 MHz, CDCl3) δ 8.31 (s, 1H, H-4), 8.16 (s, 1H, H-4'), 8.08 (s, 1H, NH), 7.89 (dd, J = 8.2, 5.4 Hz, 2H, H-7, H-2''), 7.79 (d, J = 8.5 Hz, 1H, H-6''), 7.62 (d, J = 8.6 Hz, 1H, H-6'), 7.53 (d, J = 8.8 Hz, 1H, H-6), 7.43 (d, J = 8.5 Hz, 1H, H-7'), 7.18 (d, J = 3.0 Hz, 1H, H-2'), 7.11 (t, J = 8.5 Hz, 2H, H-3'', H5''), 6.55 (d, J = 2.9 Hz, 1H, H-3'), 5.77 (s, 2H, N-CH2-O), 4.26 (t, J = 5.5 Hz, 2H, OCH2CH2Si(CH3)3), 3.94 (t, J = 5.5 Hz, 2H, N-CH2CH2-), 3.67 – 3.60 (m, 2H, N-CH2CH2-O), 0.95 – 0.89 (m, 2H, OCH2CH2Si(CH3)3), 0.84 (s, 9H, O-Si(CH3)2C(CH3)3), -0.05 (s, 9H, , OCH2CH2Si(CH3)3), -0.12 (s, 6H, O-Si(CH3)2C(CH3)3).

N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-6-yl}-1-{[2-(trimethylsilyl)ethox y]methyl}-1H-indazol-5-yl}-2,6-dichlorobenzamide (21g). Brown powder. 75.6% yield. 1H NMR (400 MHz, CD3OD) δ (ppm) 8.56 (s, 1H, H-4), 8.12 (s, 1H, H-4'), 7.78-7.64 (m, 3H, H-7, H-4'', H-6'),7.57 (d, J = 8.6 Hz, 1H, H-6), 7.52-7.39 (m, 3H, H-7', H-3'', H-5''), 7.28 (d, J = 3.1 Hz, 1H, H-2'), 6.55 (d, J = 3.1 Hz, 1H, H-3'), 5.81 (s, 2H, N-CH2-O), 4.32 (t, J = 4.8 Hz, 2H, OCH2CH2Si(CH3)3), 3.97 (t, J = 4.8 Hz, 2H, N-CH2CH2-), 3.67 (t, J = 8.0 Hz, 2H,



N-CH2CH2-O),

0.89

(t,

J

=

7.6

Hz,

2H,

OCH2CH2Si(CH3)3),

Page 34 of 63

0.79

(s,

9H,

O-Si(CH3)2C(CH3)3), -0.06 (s, 9H, OCH2CH2Si(CH3)3), -0.18 (s, 6H, O-Si(CH3)2C(CH3)3). ESI-MS m/z: 709.26 (M+H)+, calcd for C36H46Cl2N4O3Si2: 708.25.

N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-1-{[2-(trimethylsilyl)ethox y]methyl}-1H-indazol-5-yl}-3-methylbenzamide (21h). Colorless oil. 74.2% yield. 1H NMR (500 MHz, CDCl3) δ 8.37 (s, 1H), 8.18 (s, 1H, H-4)), 8.02 (s, 1H, H-4'), 7.81 (d, J = 8.5 Hz, H-7), 7.74 (s, 1H, H-2''), 7.68 (d, J = 7.9 Hz, 2H, H-6', H-6), 7.58 (d, J = 8.8 Hz, 1H, H-7'), 7.45 (d, J = 8.5 Hz, 1H, H-4''), 7.36 (d, J = 6.2 Hz, 2H, H-5'', H-6''), 7.18 (d, J = 3.1 Hz, 1H, H-2'), 6.57 (d, J = 3.0 Hz, 1H, H-3'), 5.79 (s, 2H, N-CH2-O), 4.27 (t, J = 5.5 Hz, 2H, OCH2CH2Si(CH3)3), 3.94 (t, J = 5.5 Hz, 2H, N-CH2CH2-O), 3.67 – 3.61 (m, 2H, N-CH2CH2-O), 2.43 (s, 3H, 3''-CH3), 0.96 – 0.90 (m, 2H, OCH2CH2Si(CH3)3), 0.84 (s, 9H, O-Si(CH3)2C(CH3)3), -0.05 (s, 9H, OCH2CH2Si(CH3)), -0.12 (s, 6H, O-Si(CH3)2C(CH3)3).

N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-6-yl}-1-{[2-(trimethylsilyl)ethox y]methyl}-1H-indazol-5-yl}-3,5-dimethoxybenzamide (21i). Brown powder. 83.1% yield. 1H

NMR (400 MHz, CDCl3) δ (ppm) 8.37 (s, 1H, H-4), 8.18 (s, 1H, H-4'), 7.92 (s, 1H, NH),

7.81 (dd, J = 8.5, 1.5 Hz, 1H, H-7), 7.67 (d, J = 8.6 Hz, 1H, H-6'), 7.60 (d, J = 9.0 Hz, 1H, H-6), 7.46 (d, J = 8.7 Hz, 1H, H-7'), 7.19 (d, J = 3.1 Hz, 1H, H-4''), 7.03 (d, J = 2.2 Hz, 2H, H-2'', H-6''), 6.63 (d, J = 2.2 Hz, 1H, H-2'), 6.58 (d, J = 3.1 Hz, 1H, 3'), 5.80 (s, 2H, N-CH2-O), 4.28 (t, J = 5.6 Hz, 2H, OCH2CH2Si(CH3)3), 3.95 (t, J = 6.4 Hz, 2H, N-CH2CH2-O), 3.86 (s, 6H, 2×OCH3), 3.65 (t, J = 8.0 Hz, 2H, N-CH2CH2-O), 0.91 (t, J = 7.6


Page 35 of 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hz,

2H,

OCH2CH2Si(CH3)3),

0.84

(s,

9H,

O-Si(CH3)2C(CH3)3),

-0.05

(s,

9H,

OCH2CH2Si(CH3)3), -0.12 (s, 6H, O-Si(CH3)2C(CH3)3). ESI-MS m/z: 701.42 (M+H)+, calcd for C38H52Cl2N4O5Si2: 700.34.

N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-1-{[2-(trimethylsilyl)ethox y]methyl}-1H-indazol-5-yl}picolinamide (21j). Colorless oil. 70.8% yield. 1H NMR (500 MHz, CDCl3) δ 10.19 (s, 1H, NH), 8.65 (d, J = 4.5 Hz, 1H, H-3''), 8.56 (d, J = 1.1 Hz, 1H, H-6''), 8.35 (d, J = 7.8 Hz, 1H, H-7), 8.21 (s, 1H, H-4), 7.94 (t, J = 7.8 Hz, 1H, H-6), 7.85 (ddd, J = 8.4, 4.2, 1.4 Hz, 2H, H-4'', H-5''), 7.62 (d, J = 8.9 Hz, 1H, H-6'), 7.53 – 7.45 (m, 2H, H-4', H-7'), 7.20 (d, J = 3.1 Hz, 1H, H-2'), 6.60 (d, J = 2.9 Hz, 1H, H-3'), 5.81 (s, 2H, N-CH2-O), 4.29 (t, J = 5.6 Hz, 2H, OCH2CH2Si(CH3)3), 3.96 (t, J = 5.6 Hz, 2H, N-CH2CH2-O), 3.71 – 3.61 (m, 2H, N-CH2CH2-O), 0.97 – 0.90 (m, 2H, OCH2CH2Si(CH3)3), 0.85 (s, 9H, O-Si(CH3)2C(CH3)3), -0.05 (s, 9H, OCH2CH2Si(CH3)3), -0.11 (s, 6H, O-Si(CH3)2C(CH3)3).

N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-5-yl}-1-{[2-(trimethylsilyl)ethox y]methyl}-1H-indazol-5-yl}-6-methylpicolinamide (21k). Colorless oil. 75.3% yield. 1H NMR (500 MHz, CDCl3) δ 10.27 (s, 1H, NH), 8.56 (s, 1H, H-4), 8.22 (s, 1H, H-4'), 8.15 (d, J = 7.6 Hz, 1H, H-7), 7.87 – 7.78 (m, 3H, H-4'', H-5'', H-6''), 7.62 (d, J = 8.9 Hz, 1H, H-6'), 7.48 (d, J = 8.5 Hz, 1H, H-7'), 7.35 (d, J = 7.7 Hz, 1H, H-6), 7.20 (d, J = 3.0 Hz, 1H, H-2'), 6.60 (d, J = 3.0 Hz, 1H, H-3'), 5.81 (s, 2H, N-CH2-O), 4.29 (t, J = 5.6 Hz, 2H, OCH2CH2Si(CH3)3), 3.96 (t, J = 5.6 Hz, 2H, N-CH2CH2-O), 3.69 – 3.63 (m, 2H,



Page 36 of 63

N-CH2CH2-O), 2.68 (s, 3H, 3''-CH3), 0.96 – 0.89 (m, 2H, OCH2CH2Si(CH3)3), 0.85 (s, 9H, O-Si(CH3)2C(CH3)3), -0.05 (s, 9H, OCH2CH2Si(CH3)3), -0.11 (s, 6H, O-Si(CH3)2C(CH3)3).

(E)-N-{3-{1-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-1H-indol-6-yl}-1-{[2-(trimethylsilyl)et hoxy]methyl}-1H-indazol-5-yl}-3-phenylacrylamide (21l). Yellow powder. 81.2% yield. 1H NMR (400 MHz, CDCl3) δ (ppm) 8.44 (s, 1H, H-4), 8.17 (s, 1H, H-4'), 8.07-8.00 (m, 2H, H-2'', H-6''), 7.77 (m, 3H, H-3'', H-4'', H-5''), 7.64 (dd, J = 16.4, 7.7 Hz, 2H, H-α, H-2'), 7.51-7.38 (m, 3H, H-7, H-6', H-7'), 7.17 (d, J = 3.0 Hz, 1H, H-3'), 6.61 (d, J = 15.5 Hz, 1H, H-β), 6.55 (s, 1H, H-6), 5.74 (s, 2H, N-CH2-O), 4.24 (t, J = 5.5 Hz, 2H, OCH2CH2Si(CH3)), 3.92 (t, J = 5.5 Hz, 2H, N-CH2CH2-O), 3.63 (t, J = 8.0 Hz, 2H, N-CH2CH2-O), 0.91 (t, J = 8.0 Hz,

2H,

OCH2CH2Si(CH3)3),

0.84

(s,

9H,

O-Si(CH3)2C(CH3)3),

-0.06

(s,

9H,

OCH2CH2Si(CH3)3), -0.13 (s, 6H, O-Si(CH3)2C(CH3)). ESI-MS m/z: 667.41 (M+H)+, calcd for C38H50Cl2N4O3Si2: 666.34.

General Procedure for Synthesis of 22a-l. To a solution of compound 21a-l (0. 17 mmol) and ethylenediamine (0. 17 mL, 2.59 mmol) in THF (2 mL) was added TBAF (1 M solution in THF, 2.59 mL, 2.59 mmol). The reaction mixture was heated to reflux for 24 h and then allowed to cool to room temperature. The mixture was diluted with H2O and extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO4, filtered and evaporated. The residue was purified by flash chromatography to furnish target compounds 22a-l. N-{3-[1-(2-Hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}acetamide


(22a)

.

Yellow

Page 37 of 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


powder. 38.3% yield. m.p 107.0-108.8 oC. 1H NMR (500 MHz, CD3OD) δ (ppm) 13.09 (s, 1H, 1-NH), 8.36 (s, 1H, H-4), 8.08 (s, 1H, H-4'), 7.72 (dd, J = 5.8, 3.2 Hz, 2H, H-7, H-6'), 7.64-7.55 (m, 2H, H-7', H-6), 7.32 (d, J = 3.5 Hz, 1H, H-2'), 6.55 (d, J = 3.5 Hz, 1H, H-3'), 4.32 (t, J = 7.0 Hz, 2H, NCH2CH2OH), 3.91 (t, J = 5.5 Hz, 2H, NCH2CH2OH), 2.16 ( s, 3H, NCOCH3).

13C

NMR (125 MHz, DMSO-d6) δ (ppm) 168.16 (NCOCH3), 144.82 (C-1'),

138.13 (C-3), 135.84 (C-8), 133.66 (C-8'), 130.09 (C-5), 128.36 (C-9'), 124.05 (C-2), 121.56 (C-9), 120.41 (C-4', C-6), 118.86 (C-7), 110.48 (C-6'), 110.24 (C-7'), 110.05 (C-4), 100.83 (C-3'), 60.40 (NCH2CH2OH), 48.43 (NCH2CH2OH), 23.95 (NCOCH3). HRMS (ESI): calcd for C19H19N4O2 [M+H]+: 335.1509, found: 335.1509. Purity: 96.6%, tr: 11.08 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

N-{3-[1-(2-Hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}propionamide (22b). Yellow Oil. 66.5% yield. 1H NMR (500 MHz, DMSO-d6) δ (ppm) 12.94 (s, 1H, 1-NH), 9.91 (s, 1H, NHCO), 8.44 (s, 1H, H-4), 8.04 (s, 1H, H-4'), 7.70 (d, J = 8.4 Hz, 1H, H-7), 7.61 (d, J = 8.5 Hz, 1H, H-6'), 7.52 (d, J = 8.5, 8.8 Hz, 2H, H-6, H-7'), 7.42 (d, J = 3.0 Hz, 1H, H-2'), 6.53 (d, J = 3.0 Hz, 1H, H-3'), 4.92 (s, 1H, CH2CH2OH), 4.26 (t, J = 5.5 Hz, 2H, NCH2CH2OH), 3.76 (dd, J = 10.9, 5.4 Hz, 2H, NCH2CH2OH), 2.35 (q, J = 7.5 Hz, 2H, NCOCH2CH3), 1.11 (t, J = 7.6 Hz, 3H, NCOCH2CH3). 13C NMR (125 MHz, DMSO-d6) δ (ppm) 171.68 (NCOCH2CH3), 135.60 (C-3, C-5'), 132.86 (C-8, C-8'), 129.84 (C-5), 128.32 (C-9', C-2'), 120.36 (C-9, C-6), 120.10 (C-4'), 118.47 (C-7, C-6'), 110.29 (C-7', C-4), 100.72 (C-3'), 60.35 (NCH2CH2OH), 48.38 (NCH2CH2OH), 29.45 (NCOCH2CH3), 9.72 (NCOCH2CH3). HRMS (ESI): calcd for C20H21N4O2 [M+H]+: 349.1665, found: 349.1667. Purity: 96.2%, tr: 11.27 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.



3-Chloro-N-{3-[1-(2-hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}propanamide

Page 38 of 63

(22c).

Yellow powder. 42.1% yield. m.p 60.5-62.1 oC. 1H NMR (500 MHz, DMSO-d6) δ (ppm) 13.06 (s, 1H, 1-NH), 8.06 (s, 1H, H-4), 7.86 (s, 1H, H-4'), 7.70 (d, J = 8.4 Hz, 1H, H-7), 7.68 – 7.53 (m, 3H, H-6', H-6, H-7'), 7.42 (s, 1H, H-2'), 6.53 (s, 1H, H-3'), 4.92 (s, 1H, CH2CH2OH), 4.26 (d, J = 5.0 Hz, 2H, NCH2CH2OH), 3.74 (s, 4H, NCH2CH2OH, NCOCH2CH2Cl), 3.09 (s, 2H, NCOCH2CH2Cl).

13C

NMR (125 MHz, DMSO-d6) δ (ppm)

164.28 (NCOCH2CH2Cl), (C-3, C-5', C-8), 135.67 (C-8'), 132.82 (C-5), 129.87 (C-9'), 128.40 (C-2'), 120.88 – 120.31 (C-6, C-9, C-4'), 118.67×2 (C-7, C-6'), 110.36 (C-7'), 105.90 (C-4), 100.84 (C-3'), 60.39 (NCH2CH2OH), 48.39 (NCH2CH2OH), 38.29 (NCOCH2CH2Cl), 35.61 (NCOCH2CH2Cl). HRMS (ESI): calcd for C20H20ClN4O2 [M+H]+: 383.1276, found: 383.1272. Purity: >99%, tr: 11.47 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

3-Chloro-N-{3-[1-(2-hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}benzamide

(22d).

White powder, 36.1% yield. m.p 226.3-227.7 oC. 1H NMR (500 MHz, DMSO-d6): δ (ppm) 13.04 ( s, 1H, 1-NH), 10.43 (s, 1H, NHCO), 8.55 (s, 1H, H-4), 8.09 ( s, 1H, H-2''), 8.06 ( s, 1H, H-4'), 7.97 ( d, J = 7.8 Hz, 1H, H-7), 7.79 ( d, J = 9.0 Hz, 1H, H-6''), 7.75 ( d, J = 8.4 Hz, 1H, H-6'), 7.67 (d, J = 7.8 Hz, 1H, H-6), 7.63 (d, J = 8.4 Hz, 1H, H-7'), 7.58 (t, J = 7.8 Hz, 1H, H-4''), 7.57 ( d, J = 3.0 Hz, 1H, H-5''), 7.43 ( d, J = 3.0 Hz, 1H, H-2'), 6.54 ( d, J = 3.0 Hz, 1H, H-3'), 4.93 (s, 1H, CH2CH2OH), 4.27 ( t, J = 5.4 Hz, 2H, NCH2CH2OH), 3.76 (t, J = 5.4 Hz, 2H, NCH2CH2OH).

13C

NMR (125 MHz, DMSO-d6) δ (ppm) 163.91(NHCO), 144.78

(C-5'), 138.92 (C-3), 137.07 (C-8), 135.69 (C-8'), 133.22 (C-1''), 132.27 (C-3''), 131.26 (C-4''), 130.39 (C-5''), 129.94 (C-5), 128.39 (C-9'), 127.37 (C-2''), 126.43 (C-2'), 124.87 (C-6''), 121.24 (C-9), 120.42 (C-6), 120.03 (C-4'), 118.60 (C-7), 111.74 (C-6'), 110.40 (C-7'),


Page 39 of 63 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


110.33 (C-4), 100.81 (C-3'), 60.41 (NCH2CH2OH), 48.43 (NCH2CH2OH). ESI-MS m/z: 431.1 (M+H)+, calcd for C24H19ClN4O2: 430.11. HRMS (ESI): calcd for C24H20ClN4O2 [M+H]+: 431.1276, found:431.1263. Purity: 95.6%, tr: 21.41 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

4-Chloro-N-{3-[1-(2-Hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}benzamide

(22e).

White powder, 20.5% yield. m.p 64.9-65.8 oC. 1H NMR (500 MHz, D2O) δ (ppm) 13.04 (s, 1H, 1-NH), 10.40 (s, 1H, NHCO), 8.56 (s, 1H, H-4), 8.09 (s, 1H, H-4'), 8.04 (d, J = 8.4 Hz, 2H, H-7, H-6'), 7.76 (dd, J = 8.8 Hz, 2H, H-2'', H-6''), 7.62-7.63 (m, 3H, H-6, H-7', H-3''), 7.57 (d, J = 8.9 Hz, 1H, H-5''), 7.43 (d, J = 2.7 Hz, 1H, H-2'), 6.54 (d, J = 2.5 Hz, 1H, H-3'), 4.93 (s, 1H, CH2CH2OH), 4.27 (t, J = 5.2 Hz, 2H, NCH2CH2OH), 3.76 (s, 2H, NCH2CH2OH). 13C

NMR (125 MHz, D2O) δ (ppm) 164.24 (NHCO), 136.21 (C-3, C-5'), 135.66 (C-8), 133.78

(C-8', C-4''), 132.32 (C-1''), 129.87 (C-5, C-2'', C-6''), 129.52 (C-9'), 128.37 (C-2', C-3'', C-5''), 121.23 (C-6, C-9), 120.38 (C-7, C-4'), 118.55 (C-6'), 111.67 (C-7'), 110.34 (C-4), 100.76 (C-3'), 60.37 (NCH2CH2OH), 48.39 (NCH2CH2OH). HRMS (ESI): calcd for C24H20ClN4O2 [M+H]+: 431.1276, found: 431.1280. Purity: >99%, tr: 13.11 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

4-Fluoro-N-{3-[1-(2-Hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}benzamide

(22f).

White powder, 34.2% yield. m.p 96.6-97.8 oC. 1H NMR (500 MHz, DMSO-d6) δ (ppm) 13.05 (s, 1H, 1-NH), 10.38 (s, 1H, NHCO), 8.56 (s, 1H, H-4), 8.10 (t, J = 6.9 Hz, 3H, H-4', H-7, H-6'), 7.77 (d, J = 8.7 Hz, 2H, H-2'', H-6''), 7.60 (d, J = 8.7 Hz, 2H, H-6, H-7'), 7.48 – 7.27 (m, 3H, H-3'', H-5'', H-2'), 6.54 (d, J = 2.7 Hz, 1H, H-3'), 4.95 (t, J = 5.2 Hz, 1H, CH2CH2OH),



Page 40 of 63

4.27 (t, J = 5.4 Hz, 2H, NCH2CH2OH), 3.76 (d, J = 5.3 Hz, 2H, NCH2CH2OH).

13C

NMR

(125 MHz, DMSO-d6) δ (ppm) 164.96 (NHCO), 164.26 (C-3), 162.98 (C-5'), 135.65 (C-8), 132.42 (C-8'), 131.49 (C-4''), 130.27 (C-2'', C-6''), 129.85 (C-9'), 128.35 (C-2'), 121.28 (C-6, C-9), 120.38 (C-7), 119.99 (C-4'), 118.55 (C-6'), 115.31 (C-3'', C-5''), 115.14 (s), 111.63 (C-7'), 110.33 (C-4), 100.75 (C-3'), 60.37 (NCH2CH2OH), 48.39 (NCH2CH2OH). HRMS (ESI): calcd for C24H20ClN4O2 [M+H]+: 415.1571, found: 415.1575. Purity: >99%, tr: 13.17 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

2,6-Dichloro-N-{3-[1-(2-hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl} benzamide (22g). Brown powder. 35.7% yield. m.p 156.6-157.9 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm) 13.06 ( s, 1H, 1-NH), 10.78 (s, 1H, NHCO), 8.54 ( s, 1H, H-4), 8.05 (s, 1H, H-4'), 7.72 (d, J = 8.4 Hz, 1H, H-7), 7.64 ( t, J = 10.2 Hz, 1H, H-4''), 7.63 (d, J = 9.0 Hz, 1H, H-6'), 7.59 ( d, J = 8.4 Hz, 1H, H-6), 7.57 ( d, J = 8.4 Hz, 2H, H-3'', H-5''), 7.51 ( d, J = 7.2 Hz, 1H, H-7'), 7.42 ( d, J = 3.0 Hz, 1H, H-2'), 6.53 ( d, J = 3.0 Hz, 1H, H-3'), 4.27 ( t, J = 5.4 Hz, 2H, NCH2CH2OH), 3.76 (t, J = 5.4 Hz, 2H, NCH2CH2OH).

13C

NMR (150 MHz, DMSO-d6) δ

(ppm) 161.89 (NHCO), 144.83 (C-5', C-3), 138.89 (C-8), 136.54 (C-8'), 135.70 (C-1''), 132.15 (C-2'', C-6''), 131.32 (C-4''), 129.99 (C-5), 128.39 (C-9'), 128.25 (C-3'', C-5'', C-2'), 124.80 (C-9), 120.42 (C-6), 120.08 (C-4'), 118.60 (C-7, C-6'), 110.75 (C-7'), 110.44 (C-4, C-3'), 60.41 (NCH2CH2OH), 48.43 (NCH2CH2OH). HRMS (ESI): calcd for C24H20ClN4O2 [M+H]+: 465.0886, found: 465.0889. Purity: 99.1%, tr: 19.64 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

N-{3-[1-(2-Hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}-3-methylbenzamide


(22h).

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White powder, 45.2% yield. m.p 72.8-73.5 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm) 13.02 (s, 1-NH), 10.29 (s, 1 NHCO), 8.56 (s, 1H, H-4), 8.09 (s, 1H, H-4'), 7.82-7.75 (m, 4H, -7, H-6', H-6, H-2''), 7.62 (d, J = 8.5 Hz, 1H, H-7'), 7.55 (d, J = 8.8 Hz, 1H, H-6''), 7.42 (d, J = 6.3 Hz, 3H, H-4'', H-5'', H-2'), 6.53 (s, 1H, H-3'), 4.93 (s, 1H, NCH2CH2OH), 4.27 (s, 2H, NCH2CH2OH), 3.76 (d, J = 4.9 Hz, 2H, NCH2CH2OH), 2.41 (s, 3H, Ph-CH3). 13C NMR (125 MHz, DMSO-d6) δ (ppm) 165.47 (NHCO), 137.62 (C-5', C-3), 135.65 (C-8), 135.11 (C-8', C-3''), 132.60 (C1''), 131.93 (C-4''), 129.87 (C-2''), 129.61 (C-5), 128.36 (C-9', C-5''), 128.22 (C-2'), 128.06 (C-6''), 124.73 (C-9), 121.23 (C-6), 120.40 (C-4'), 118.55 (C-7), 111.47 (C-6'), 110.34 (C-7'), 110.21 (C-4), 100.77 (C-3'), 60.38 (NCH2CH2OH), 48.41 (NCH2CH2OH), 20.96 (-CH3). HRMS (ESI): calcd for C25H23N4O2 [M+H]+: 411.1822, found: 411.1827. Purity: >99%, tr: 13.34 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

N-{3-[1-(2-Hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}-3,5-dimethoxy

benzamide

(22i). Brown powder. 34.7% yield. m.p 121.8-123.9 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm) 13.05 (s, 1H, 1-NH), 10.29 (s, 1H, NHCO), 8.53 ( s, 1H, H-4), 8.05 (s, 1H, H-4'), 7.72 (d, J = 9.0 Hz, 2H, H-7, H-6'), 7.59 ( d, J = 8.5 Hz, 1H, H-7'), 7.52 ( d, J = 9.0 Hz, 1H, H-6), 7.39 (d, J = 3.0 Hz, 1H, H-2'), 7.14 (d, J = 1.8 Hz, 2H, H-3'', H-5''), 6.67 ( s, 1H, H-4''), 6.50 (d, J = 3.0 Hz, 1H, H-3'), 4.95 ( s, 1H, NCH2CH2OH), 4.23 (t, J = 5.4 Hz, 2H, NCH2CH2OH), 3.80 ( s, 6H, 2×OCH3), 3.72 ( t, J = 5.4 Hz, 2H, NCH2CH2OH).

13C

NMR (150 MHz,

DMSO-d6) δ (ppm) 164.97 (NHCO), 160.45 (C-2'', C-6''), 144.76 (C-5', C-3), 138.94 (C-8), 137.19 (C-8'), 135.74 (C-4''), 132.47 (C-5), 129.95 (C-9'), 128.45 (C-2'), 124.98 (C-9), 121.48 (C-6), 120.48 (C-4'), 120.03 (C-7), 118.64 (C-6'), 111.83 (C-7'), 110.43 (C-3'', C-5''), (C-4) ,



Page 42 of 63

105.68 (C-4), 103.37 (C-1''), 100.86 (C-3'), 60.46 (NCH2CH2OH), 55.59 (OCH3), 48.48 (NCH2CH2OH). HRMS (ESI): calcd for C26H24N4O4 [M+H]+: 457.1877, found: 457.1877. Purity: 97.3%, tr: 21.38 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

N-{3-[1-(2-Hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}picolinamide (22j). Red oil. 50.3% yield. 1H NMR (600 MHz, DMSO-d6) δ (ppm) 13.05 (s, 1H, 1-NH), 10.76 (s, 1H, NHCO), 8.75 (dd, J = 8.1, 2.5 Hz, 2H, H-3'', H-6''), 8.19 (d, J = 7.8 Hz, 1H, H-7), 8.12 (s, 1H, H-4), 8.07 (td, J = 7.7, 1.6 Hz, 1H, H-5''), 7.90 (d, J = 8.9 Hz, 1H, H-6'), 7.77 (d, J = 8.5 Hz, 1H, H-6), 7.72 – 7.65 (m, 1H, H-4''), 7.63 (d, J = 8.6 Hz, 1H, H-7'), 7.57 (s, 1H, H-4'), 7.42 (d, J = 3.0 Hz, 1H, H-2'), 6.55 (d, J = 2.8 Hz, 1H, H-3'), 4.99 (t, J = 5.2 Hz, 1H, NCH2CH2OH), 4.27 (t, J = 5.5 Hz, 2H, NCH2CH2OH), 3.76 (d, J = NCH2CH2OH).

13C

5.5 Hz, 2H,

NMR (125 MHz, DMSO-d6) δ (ppm) 162.50 (NHCO), 150.18 (C-1''),

148.51 (C-3, C-5', C-3''), 138.19 (C-8), 135.79 (C-5'', C-8'), 131.88 (C-5), 129.98 (C-9'), 128.51 (C-4''), 126.87 (C-2'), 122.36 (C-9), 121.16 (C-6), 120.53 (C-6'), 118.77 (C-4', C-7, C-6''), 111.61 (C-7'), 110.46 (C-4), 100.96 (C-3'), 60.50 (NCH2CH2OH), 48.50 (NCH2CH2OH). HRMS (ESI): calcd for C23H20N5O2 [M+H]+: 398.1618, found: 398.1617. Purity: 95.2%, tr: 11.96 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

N-{3-[1-(2-Hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}-6-methylpicolinamide

(22k).

Red powder. 60.1% yield. m.p 146.7-148.1 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm) 12.89 (s, 1H, 1-NH), 10.41 (s, 1H, NHCO), 8.54 (s, 1H, H-4), 7.97 (s, 1H, H-4'), 7.82-7.79 (m, 2H, H-7, H-6'), 7.70 (d, J = 8.7 Hz, 1H, H-6''), 7.63-7.60 (m, 1H, H-5''), 7.48 (d, J = 8.5 Hz,


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1H, H-4''), 7.42 (d, J = 8.8 Hz, 1H, H-6), 7.38 (d, J = 7.3 Hz, 1H, H-7'), 7.27 (d, J = 2.7 Hz, 1H, H-2'), 6.40 (d, J = 2.7 Hz, 1H, H-3'), 4.79 (t, J = 5.2 Hz, 1H, NCH2CH2OH), 4.12 (t, J = 5.4 Hz, 2H, NCH2CH2OH) , 3.61 (dd, J = 10.6, 5.2 Hz, 2H, NCH2CH2OH), 2.35 (s, 3H, -CH3).

13C

NMR (125 MHz, DMSO-d6) δ (ppm) 162.42

(NHCO), 157.23 (C-3''), 149.38

(C-1''), 144.81 (C-3, C-5'), 138.91 (C-8), 138.11 (C-5''), 135.67 (C-8'), 129.85 (C-5), 128.38 (C-9'), 126,28 (C-4'', C-2'), 121.06 (C-9, C-6), 120.43 (C-6'), 119.27 (C-6''), 118.64 (C-4', C-7), 110.34 (C-7', C-4), 100.83 (C-3'), 60.38 (NCH2CH2OH), 48.39 (NCH2CH2OH), 23.81 (-CH3). HRMS (ESI): calcd for C24H22N5O2 [M+H]+: 398.1618, found: 398.1617. Purity: 95.6%, tr: 23.49 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

N-{3-[1-(2-Hydroxyethyl)-1H-indol-5-yl]-1H-indazol-5-yl}cinnamamide

(22l).

Yellow

powder. 45.6% yield. m.p 207.7-210.4 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm) 13.01 (s, 1H, 1-NH), 10.32 (s,1H, NHCO), 8.62 ( s, 1H, H-4), 8.08 ( s, 1H, H-4'), 7.74 (d, J = 8.5 Hz, 1H, H-7), 7.70-7.61 (m, 5H, H-6', H-7', H-2'', H-6'', H-4''), 7.56 (d, J = 9.0 Hz, 1H, H-6), 7.49-7.41 (m, 4H, H-3'', H-5'', H-α, H-2'), 6.88 (d, J = 18.4 Hz, 1H, H-β), 6.54 (d, J = 2.4 Hz, 1H, H-3'), 4.28 (t, J = 6.0 Hz, 2H, NCH2CH2OH), 3.77 (t, J = 5.4 Hz, 2H, NCH2CH2OH). 13C NMR (150 MHz, DMSO-d6) δ (ppm) 163.41 (NHCO), 144.71 (C-3, C-5'), 139.77 (NHCH=CH-), 138.65 (C-8), 135.68 (C-8'), 134.85 (C-1''), 132.81 (C-5), 129.96 (C-9'), 129.70 (C-2'', C-6''), 129.05×2 (C-3'', C-5''), 128.40 (C-4''), 127.69 (C-2''), 124.93 (C-9), 122.49

(C-6), 120.43 (C-4'), 120.16 (NHCH=CH-), 120.10 (C-7), 118.58 (C-6'), 110.61

(C-7'), 110.42 (C-4), 110.75 (C-3'), 60.43 (NCH2CH2OH), 48.45 (NCH2CH2OH). HRMS (ESI): calcd for C26H23N4O2 [M+H]+: 423.1822, found: 423.1825. Purity: 94.5%, tr: 8.61 min.,



Page 44 of 63

eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

N-[3-(1H-Indol-5-yl)-1H-indazol-5-yl]-2,6-dichlorobenzamide (22m). Compound 22m was made in a manner similar to compounds 22a-l. oC. 1H

White solid. 34.2% yield. m.p 192.4-194.7

NMR (600 MHz, DMSO-d6) δ (ppm) 11.23 (s, 1H, 1-NH), 10.79 (s, 1H, NHCO), 8.54

(s, 1H, 1'-NH), 8.06 (s, 1H, H-4), 7.78-7.47 (m, 8H, H-4', H-7, H-6', H-6, H-7', H-3'', H-4'', H-5''), 7.40 (s, 1H, H-2'), 6.53 (s, 1H, H-3'). 13C NMR (150 MHz, DMSO-d6) δ (ppm) 170.44 (NHCO), 161.95 (C-5'), 136.64 (C-3), 135.68 (C-8), 132.24

(C-8'), 131.40 (C-1'), 128.76

(C-2'', C6''), 128.34 (C-4''), 128.00 (C-3'', C-5''), 126.18 (C-5), 124.81 (C-9'), 120.65 (C-2'), 120.13 (C-9), 120.13 (C-6), 118.42 (C-4'), 111.93 (C-7), 110.92 (C-6'), 110.49 (C-7'), 101.58 (C-3'). HRMS (ESI): calcd for C22H14Cl2N4O [M+H]+: 421.0620, found: 421.0628. Purity: 95.3%, tr: 20.26 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

N-[1-Benzyl-3-(1H-indol-5-yl)-1H-indazol-5-yl]-2,6-dichlorobenzamide (22n). Compound 22n was made in a manner similar to compounds 21a-l. White oil. 60.9% yield. 1H NMR (600 MHz, DMSO-d6) δ (ppm) 11.24 (s, 1H, 1'-NH), 10.83 (s, 1H, NHCO), 8.57 (s, 1H, NH2), 8.07 (s, 1H, H-4), 7.76 (d, J = 6.0 Hz, 1H, H-7), 7.69-7.66 (m, 2H, H-6', H-4''), 7.60 (s, 1H, H-4'), 7.58 (s, 1H, NH2), 7.56 (d, J = 6.0 Hz, 1H, H-6), 7.51 (dd, J = 8.6, 7.6 Hz, 1H, H-7'), 7.51 (dd, J = 9.0, 7.3 Hz, 1H, H-3'', 5''), 7.42 (t, J = 2.4 Hz, 1H, H-2'), 7.35-7.23 (m, 5H, Bn-H2'', Bn-H3'', Bn-H4'', Bn-H5'', Bn-H6''), 6.55 (s, 1H, H-3'), 5.71 (s, 2H, N-CH2-Bn).

13C

NMR

(150 MHz, DMSO-d6) δ (ppm) 162.00 (NHCO), 144.52 (C-3, C-5'), 138.34 (C-8), 137.73 (Bn-C1), 136.55 (C-8'), 135.76(C-1''), 131.44 (C-2'', C-6''), 131.37 (C-4''), 128.65 (C-5),


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128.33 (C-3'', C-5''), 128.02 (Bn-C2, Bn-C6), 127.57 (Bn-C4), 124.27 (C-2'), 121.02 (C-4), 120.64 (C-6), 120.28 (C-4'), 118.55 (C-6'), 111.98 (C-7'), 110.72 (C-7), 110.60 (C-4), 101.63 (C-3'), 59.84 (N-CH2-Bn). HRMS (ESI): calcd for C29H21Cl2N4O [M+H]+: 511.1094, found: 511.1087. Purity: 95.1%, tr: 13.05 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

2-[5-(5-Nitro-1H-indazol-3-yl)-1H-indol-1-yl] ethanol (22o). Compound 22o was made in a manner similar to compounds 22a-l. Yellow powder, 71.9% yield. m.p 184.1-185.9 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm) 13.74 (s, 1H, 1'-NH), 8.94 (s, 1H, H-4), 8.25 (d, J = 10.0 Hz, 1H, H-7), 8.18 (s, 1H, H-4'), 7.77 (d, J = 10.0 Hz, 1H, H-6), 7.68 (d, J = 6.0 Hz, 1H, H-6'), 7.46 (d, J = 3.0 Hz, 2H, H-7', H-2'), 6.61 (d, J = 3.0 Hz, 1H, H-3'), 4.28 (t, J = 5.5 Hz, 2H, NCH2CH2OH), 3.76 (t, J = 4.5 Hz, 2H, NCH2CH2OH). 13C NMR (150 MHz, DMSO-d6) δ (ppm) 143.56 (C-8), 142.91 (C-5', C-3), 141.76 (C-5), 136.75 (C-8'), 131.39 (C-9'), 129.65 (C-2'), 129.54 (C-4), 121.72 (C-6), 120.62 (C-4'), 119.42 (C-4), 111.79 (C-7, C-7'), 102.47 (C-3'), 61.54 (NCH2CH2OH), 49.17 (NCH2CH2OH). HRMS (ESI): calcd for C17H13N4O3 [M-H]+: 321.0987, found: 321.0987. Purity: 96.1%, tr: 14.54 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

2-[5-(5-Amino-1H-indazol-3-yl)-1H-indol-1-yl] ethanol (22p). Compound 22p was made in a manner similar to compound 20. Black powder. 60.9% yield. m.p 153.0-155.7 oC. 1H NMR (600 MHz, DMSO-d6) δ (ppm) 12.68 (s, 1H, 1'-NH), 8.02 (s, 1H, H-4'), 7.70 (d, J = 8.7 Hz, 1H, H-7), 7.56 (d, J = 8.6 Hz, 1H, H-6'), 7.39 (d, J = 3.0 Hz, 1H, H-2'), 7.28 (d, J = 8.7 Hz, 1H, H-6), 7.17 (s, 1H, H-3'), 6.81 (d, J = 8.6, 1H, H-7'), 4.86 (s, 2H, NH2), 4.25 (t, J = 5.5 Hz,



Page 46 of 63

2H, NCH2CH2OH), 3.75 (t, J = 5.6 Hz, 2H, NCH2CH2OH). 13C NMR (150 MHz, DMSO-d6) δ (ppm) 142.72 (C-5', C-3), 141.45 (C-5), 135.34 (C-8', C-8),

129.63 (C-9'), 128.30 (C-2'),

126.61 (C-9), 121.16 (C-4'), 120.26 (C-7), 118.10 (C-6), 117.84 (C-6'), 110.12 (C-7'), 100.64 (C-4, C-3'), 60.37 (NCH2CH2OH), 48.37 (NCH2CH2OH). HRMS (ESI): calcd for C17H15N4O [M-H]+: 292.1245, found: 292.1248. Purity: 95.2%, tr: 6.88 min., eluent: MeOH/ H2O 40/60 to 10/90, fl: 1.0 mL/min.

3-(1H-Indol-5-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-indazol-5-amine

(23).

Compound 23 was made in a manner similar to compound 20. Brown oil, 71.9% yield. 1H NMR (500 MHz, CDCl3) δ (ppm) 8.40 (s, 1H, H-4), 8.15 (s, 1H, H-4'), 7.75 (d, J = 10.0 Hz, 1H, H-7), 7.46 (d, J = 5.0 Hz, 1H, H-2'), 7.41 (d, J = 10.0 Hz, 1H, H-6'), 7.33 (s, 1H, H-6), 7.22 (s, 1H, NH2), 6.93 (d, J = 10.0 Hz, 1H, H-7'), 6.62 (s, 1H, H-3'), 5.73 (s, 2H, N-CH2-O), 3.63 (t, J = 10.0 Hz, 2H, OCH2CH2Si(CH3)3), 0.91 (t, J = 10.0 Hz, 2H, OCH2CH2Si(CH3)3), -0.06 (s, 9H, OCH2CH2Si(CH3)3). 13C NMR (125 MHz, CDCl3) δ (ppm) 144.92 (C-3, C-5'), 140.58 (C-5), 136.89 (C-8'), 135.67 (C-8), 128.22 (C-9'), 125.53 (C-2'), 124.73 (C-9), 123.72 (C-4'), 122.09 (C-6), 119.80 (C-6'), 118.48 (C-7'), 111.34 (C-7), 110.54 (C-3'), 103.04 (C-4), 77.75

(NCH2O),

66.22

(OCH2CH2Si(CH3)3),

17.81

(OCH2CH2Si(CH3)3),

-1.46

(OCH2CH2Si(CH3)3). ESI-MS m/z: 379.2 (M+H)+, calcd for C21H26N4OSi: 378.5.

1-Benzyl-3-(1H-indol-5-yl)-1H-indazol-5-amine (24). Compound 24 was made in a manner similar to compound 20. Brown oil. 1H NMR (600 MHz, DMSO-d6) δ (ppm) 11.16 (s, 1H, 1'-NH), 8.04 (s, 1H, H-4'), 7.66 (d, J = 6.0 Hz, 1H, H-7), 7.49 (d, J = 12.0 Hz, 1H, H-6'), 7.41


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(d, J = 12.0 Hz, 1H, H-7')), 7.38 (s, 1H, H-4), 7.34-7.22 (m, 5H, Bn-H2'', Bn-H3'', Bn-H4'', Bn-H5'', Bn-H6'' ), 7.19 (s, 1H, H-2'), 6.83 (d, J = 6.0 Hz, 1H, H-6), 6.51 (s, 1H, H-3'), 5.57 (s, 2H, N-CH2-Bn), 4.92 (s, 2H, NH2).

13C

NMR (150 MHz, DMSO-d6) δ (ppm) 143.34

(C-3), 142.25 (C-5'), 138.13 (C-5), 135.64 (Bn-C1), 135.39 (C-8), 128.54 (Bn-C3, Bn-C5, C-9')),

127.95 (Bn-C4), 127.38 (Bn-C2, Bn-C6), 125.94 (C-2'), 125.28 (C-9), 122.28 (C-4'),

120.52 (C-6), 118.08 (C-6'), 117.97 (C-7'), 110.72 (C-7), 110.43 (C-3'), 101.53 (C-4), 59.84 (N-CH2-Bn). ESI-MS m/z: 339.2(M+H)+, calcd for C22H18N4: 339.5.

Cells and Biological Reagents. Mouse IL-6 and TNF-α enzyme-linked immunosorbent assay (ELISA)

kits

were

obtained

from

eBioscience

Inc.

(San

Diego,

CA,

USA).

Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (St Louis, MO, USA). Recombinant MD2, TLR4, and MyD88 proteins were obtained from R&D Company (Minneapolis, MN, USA). MD2 antibody was from eBioscience (San Diego, CA, USA). Antibodies for p65, Lamin B, and TLR4 were from Santa Cruz Biotechnology (Dallas, TX). Tumor necrosis factor-α (TNF-α) and F4/80 antibodies were obtained from Abcam (Cambride, MA, USA). Mouse primary peritoneal macrophages (MPMs) were prepared from Institute of Cancer Research (ICR) mice using the method described in our previous study.52 MPMs were cultured in DMEM media (Gibco, Eggenstein, Germany) supplemented with 10% FBS (Hyclone, Logan, UT), 100mU/ml penicillin, and 100 mg/mL streptomycin. Compounds were added into cell cultural medium in DMSO solution with the final 0.1% concentration of DMSO.



Enzyme-Linked Immunosorbent Assay (ELISA). After treatment with indicated compounds and LPS, the levels of cytokines IL-6 and TNF-α in cell medium were determined by an ELISA kit according to the instructions of manufacturer. Briefly, MPMs were pretreated with compounds for 2 h before exposure to 0.5 μg/mL LPS. After 22 h, the culture media and cells were collected separately. The total proteins from cells were determined using Bio-Rad protein assay. Amount of the inflammatory factors in culture media was normalized to the total protein amount obtained from cell pellets.

MTT Assay. MPMs were seeded into 96-well plates at a concentration of 2.0 ×104 cells per well in DMEM media, supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. After treatment with the compounds at indicated concentrations for 24h, a fresh solution of MTT (5 mg/ml) prepared in NaCl solution (0.9%) was added to each well and the plate was then incubated in 5% CO2 at 37 oC for 4 h. Absorbance at 490 nm was detected by a Bio-Rad multi-well-plate reader.

NF-κB Reporter Assay. To measure NF-κB activity, we prepared NF-κB-EGFP RAW264.7 stable cell line by lentivirus infection. Particles containing the response element of NF-κB was first generated by co-transfecting 293T cells with p-LV-NFκB-RE-EGFP (Inovogen) and packaging plasmids (psPAX2 and pMD2.G) using PEI (Sigma). Supernatant was collected after 48 h and filtered using a 0.45 μm filter. RAW264.7 cells (ATCC; Manassas, VA) were incubated with the supernatant and 8 μg/mL polybrene (Sigma) for 12 h. Stable cells were selected with 2 μg/mL puromycin (Invitrogen, San Diego). NFκB-EGFP RAW264.7 cells were then treated with 500 ng/mL LPS for 1h, followed by vehicle or 22m (10 μM) treatment


Page 48 of 63

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for 3 h. The EGFP signal was detected by flow cytometry.

Kinase Inhibition Profiling. The kinase selectivity profile was tested on the DiscoverX KINOMEscan platform (http://www.kinomescan.com/) in DiscoverX Corporation (Fremont, CA). Compound 22m was screened at 1 μM. Results of the primary screen are reported as percent of control (% Ctrl), where lower numbers indicate stronger hits. The sreened kinases and inhibition rates were shown in Supplementary Table S1. The % Ctrl was calculated as follows: % control = [(test compound signal − positive control signal)/(negative control signal − positive control signal)] × 100. Negative control = DMSO (100%Ctrl). Positive control = control compound (0%Ctrl).

Surface Plasmon Resonance (SPR) Analysis. SPR biosensing experiments were performed at 22 °C on Biacore T200 instruments equipped with CM5 sensor chips (GE healthcare). Briefly, MD2 or TLR4 protein (in acetate acid buffer) was immobilized on the sensor after activation by 40 mM EDC and 10 mM sNHS in water solution. Then 22m at different concentrations was dissolved in running buffer (PBS, 0.1% SDS and 0.05% Tween-20) with added 5% DMSO. Compounds at different concentrations were injected simultaneously. Data processing and analysis were performed using the Biacore 4000 and Biacore T200 evaluation software (GE healthcare).

Immunoprecipitation. Macrophages were stimulated with 0.5 µg/mL LPS for 15 min after a 30-minute pretreatment with 22m at 10 μM. Cells were lysed with buffer containing protease



and phosphatase inhibitors. Cell extracts were incubated with adequate amount of anti-MD2 antibody and precipitated with protein A+G agarose beads (Beyotime Biotech, Nantong, China) at 4°C overnight. After boiling, the released proteins were detected by immunoblot using anti-TLR4 antibody. HA- and Flag-tagged TLR4 plasmids were constructed and sequenced in HEK293T cells. Cells were transfected with two plasmids using Lipofectamine 3000 (Invitrogen). Six hours later, cells were treated with different concentrations of 22m or vehicle. Cell lysates were collected for immunoprecipitation using protein A/G agarose beads. Immunocomplexes captured were detected by immunoblot using anti-HA and anti-flag antibodies from Santa Cruz Biotechnology (Dallas, TX).

Flow Cytometric Analysis of LPS Binding. Binding of fluorescein isothiocyanate-labeled LPS (LPS-FITC, from E. coli 055:B5, Sigma, St. Louis, MO) to cell membranes was measured. Briefly, MPMs were seeded at 1 × 106 and incubated with LPS-FITC (50 μg/ mL) for 30 min with or without 22m. After washing, bound LPS-FITC was examined by flow cytometry.

Molecular Docking of Compounds to the MD2/TLR4 Structural Model. The crystal structure of TLR4-MD-2 complex was obtained from Protein Data Bank (PDB ID: 3FXI). Water molecules and original ligand was manually removed by using PyMol software.53 The predicted binding pose of compound 22m in the dimerization surface was carried out using Autodock (version 4.2.6).54 Prepare_ligand4.py and prepare_recptor4.py scripts from


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AutoDockTools 1.5.6 were used to prepare the initial files including adding charges and hydrogen atoms. Then, a grid box of 60 × 60 × 60 with a spacing of 0.375Å enclosed the whole binding site. The Lamarckian genetic (LGA) was adopted to search for the best binding poses. The specific docking settings were as follow: trials of 100 dockings, 300 individuals per population with a crossover rate of 0.8 and the local search rate was set to 0.06. Other parameters were set as default during the docking.

Animal Model of ALI. Male C57BL/6 mice and Institute of Cancer Research (ICR) mice weighing approximately 18 to 22 g were obtained from the Animal Center of Wenzhou Medical University (Wenzhou, China). The animals were acclimatized in an air-conditioned room maintained in a 12:12-h light/dark cycle and fed standard chow and water. Mice used in this study were treated in accordance with the Guide for Care and Use of Laboratory Animals of National Institutes of Health. The present study was approved by Wenzhou Medical College Animal Policy and Welfare Committee. Compounds 22m and X12 were dissolved with 0.1% sodium carboxyl methyl cellulose (CMC-Na). Male C57BL/6 mice weighing 18−22 g were randomly divided into 5 goups (n=8 per group): control group, LPS group, 22m (5 mg/kg) group, 22m (10 mg/kg) group and X12 (10 mg/kg) group. Mice were treated with compounds’ solution by tail vein injection 15 min before an intraperitoneal injection of LPS (20 mg/kg). Control group animals were received only an equal volume of saline. Mice were anesthetized and sacrificed 6 h after LPS injection. Blood samples were collected from the right ventricle orbital veniplex using a heparinized syringe with a needle. Lung tissues were harvested. ICR mice were used for MPM preparation



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as indicated above.

Histopathologic Examination of Lung Tissues. The superior lobe of right lung was collected and fixed in 4% paraformaldehyde solution. Tissues were then embedded in paraffin and cut into 5 μm sections. After dehydration, the sections were stained with hematoxylin and eosin (H&E) using standard protocol and observed with a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan). To determine the lung injury score, images were evaluated by an treatment-blinded investigator as previously described30. Each lung injury index were measured using the following four categories: alveolar congestion, hemorrhage, neutrophil infiltration into the airspace or vessel wall, and thickness of alveolar wall/ hyaline membrane formation. Each category was graded on a 0 to 4-point scale: 0 = no injury, 1 = injury up to 25% of the field, 2 = injury up to 50% of the field, 3 = injury up to 75% of the field, and 4 = diffuse injury.

Measurement of Myeloperoxidase (MPO) Activity. For MPO activity, tissue lysates were incubated with 0.01% H2O2 in the presence of O-dianisidine dihydrochloride (0.167 mg/mL) for 30 min. The change in absorbance at 460 nm for each sample was recorded by a plate reader (Bio-Tek Instruments Inc.). MPO activity was characterized as the quantity of enzyme degrading 1 μM peroxide/min at 37 °C and is expressed in units per gram lung tissue.

Lung Immunohistochemistry. Immunohistochemistry was performed using an ABC staining kit (Vector Laboratories). Tissue sections were deparaffinized in xylene and hydrated


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using an ethanol gradient. After blocking endogenous peroxidase activity, antigen retrieval was performed by heating slides in a microwave oven in 0.1 M citrate-buffered solution (pH 6.0) for 10 min. All sections were blocked in 5% bovine serum albumin (BSA) and incubated with primary anti-F4/80 antibody overnight at 4 °C. The slides were then incubated with HRP-labeled secondary antibody for 10 min. Immunoreactivity was detected using 3,3-diaminobenzidine (DAB). Slides were counterstained with hematoxylin. The percentage of F4/80-positive inflammatory cells was calculated in 10 randomly selected fields (at 200×) per section using ImageJ (https://imagej.nih.gov/ij/).

Real-Time Quantitative PCR. Total RNA was extracted from the lung tissue using RNeasy kit (Takara, Shiga, Japan) and residual DNA was removed by DNase I (Takara). Both reverse transcription and quantitative PCR were conducted using a two-step M-MLV Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen). Eppendorf Mastercycler eprealplex detection system (Eppendorf, Hamburg, Germany) was used for q-PCR. The primers of genes including TNF-α, IL-6, IL-1β, COX-2, IL-12, IL-33 and β-actin were obtained from Invitrogen. The amount of each gene was determined and normalized by the amount of β-actin. The primer sequences of mouse genes are shown in Supplementary Table S2.

Statistical Analysis. Results are presented as the mean ± standard error of mean (SEM). Student's t test or ANOVA multiple comparisons were used to test for differences between sets of data. Analyses were performed using GraphPad Pro (GraphPad, San Diego, CA). P values less than 0.05 were considered significant. All experiments were repeated at least three times. ACS Paragon Plus Environment


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AUTHOR INFORMATION Corresponding author *Z.L.: E-mail: [email protected]; phone, +86-577-86699892. *G.L.: E-mail: [email protected]; phone, +86-577-86699396.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was supported by the National Key Research and Development Program of China (2017YFA0506000), the National Natural Science Funding of China (81773579, 81622043, 81770825, and 21572167), and the Natural Science Funding of Zhejiang Province (LY17B020008).

Abbreviations Used: COX-2, cyclooxygenase 2; DMF, N,N-dimethylformamide; DMSO, dimethyl sulphoxide; ELISA, enzyme linked immunosorbent assay; IL-6, Interleukin-6; IKKβ, inhibitor kappa B kinaseβ; JNK, c-Jun N-terminal kinase; LOS, lipooligosaccharide; LPS, lipopolysaccharide; MD2, myeloid differentiation protein-2; MyD88, myeloid differentiating primary response gene 88; MPMs, mouse peritoneal macrophages; MPO, myeloperoxidase; Na2S2O3, Sodium thiosulfate;

NaH,

sodium

hydride;

NF-κB,

nuclear

factor-kappa

B;

PAMPs,

pathogen-associated molecular patterns; Pd/C, palladium/carbon; PRRs, pattern recognition


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receptors; SAR, structure-activity relationship; TBS, tert-butyldimethylsilyl; TNF-α, tumor necrosis factor alpha; TLR4, toll-like receptor 4;

Ancillary Information Supporting Information. Figure S1, Table S1 and Table S2, and the 1H NMR, 13C NMR, and HPLC spectra data of synthesized compounds (PDF), Molecular-formula strings (CSV).



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Table of Contents Graphic


Discovery of 3-(Indol-5-yl)-indazole Derivatives as Novel Myeloid

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